Serine/threonine kinase and nucleic acids encoding same

ABSTRACT

A serine/threonine kinase protein which is associated with mitotic and meiotic cell division and which is characterized by having a kinase domain in its N-terminus and three PEST regions in the C-terminus, and nucleic acid molecules encoding the protein. Diagnostic and therapeutic methods using the serine/threonine kinase protein and nucleic acid molecules are also described.

FIELD OF THE INVENTION

The invention relates to a novel serine/threonine kinase protein andisoforms and parts thereof, nucleic acid molecules encoding the novelprotein and fragments thereof, and uses of the protein and nucleic acidmolecules.

BACKGROUND OF THE INVENTION

The Drosophila polo and Saccharomyces cerevisiae CDC5 genes encodeserine/threonine kinases which are highly related in sequence (47%identity in the catalytic domain) and in function (Llamazares, S. etal., (1991) Genes Dev. 5,2153-2165, and Kitada, K. et al, (1993) Mol.Cell. Biol. 13, 4445-4457). Phenotypic analysis of mutant alleles ofpolo and CDC5 indicates that both are required for the proper formationand function of the spindle during mitotic and meiotic cell divisions(Llamazares, S. et al., (1991) Genes Dev. 5, 2153-2165, 1991; Fenton, B.& Glover, D. M. (1993) Nature 363, 637-640; Sharon, G. & Simchen, G.(1990) Genet. 125, 475-485; Schild, D. & Byers, B. (1980) Genet. 96,859-876). Two related murine genes, snk (Simmons, D. L., Neel, B. G.,Stevens, R., Evett, G. & Erikson, R. L. (1992) Mol. and Cell. Biol. 12,4164-4169) and plk (Clay, F. J., McEwen, S. J., Bertoncello, I., Wilks,A. F. & Dunn, A. R. (1993) Proc. Natl. Acad. Sci. USA 90, 4882-4886)have recently been cloned and may also play a role in regulating cellproliferation based on cell cycle dependent changes in theirtranscription levels and a correlation between the tissue specificpattern of plk expression and active cell division.

SUMMARY OF THE INVENTION

The present inventors have identified and characterized aserine/threonine kinase protein that plays an important role in cellproliferation. In particular, the present inventors isolated murinecDNAs encoding isoforms of a serine/threonine kinase, designated Sak.The isoforms, Sak-a and Sak-b, differ in their non-catalytic C-terminalends. Sak is related to members of the polo subfamily of kinase. Sak hasa kinase domain in the N-terminal region (See FIG. 1) and the sequencein the N-terminal region has significant homology to the polo subfamily.The sak kinase domain also diverges significantly from other members ofthe polo subfamily. Within subdomain VI-B, the threonine residue in thesak sequence DLTLSN is in a position normally occupied by a lysine, notonly in proteins within the polo subfamily, but in the majority ofserine/threonine kinases. The serine residue in this sequence isreplaced by a glycine in all other members of the polo subfamily. A 30amino acid homology domain identified in the C-terminus of the polo,CDC5, plk and snk kinases is absent in both of the sak proteins (Clay,F. J., McEwen, S. J., Bertoncello, I., Wilks, A. F. & Dunn, A. R. (1993)Proc.-b Natl. Acad. Sci. USA 90, 4882-4886). Three PEST regions, eachcontaining 2 sequences in tandem in Sak-a and one in Sak-b, were alsoidentified in the C-terminal region Of the proteins.

Northern and in situ RNA analyses of sak expression in mouse embryos andadult tissues revealed that expression is associated with mitotic andmeiotic cell division. In addition, during embryogenesis, sak expressionis prominent in the respiratory and olfactory mucosa. The pattern of sakexpression and its sequence homology with the polo gene family indicatethat sak may play a role in cell proliferation. In support of this, thepresent inventors found that cell growth was suppressed by expression ofa sak-a antisense fragment in CHO cells.

The present invention therefore provides a purified and isolated nucleicacid molecule containing a sequence encoding a serine/threonine kinaseprotein which is associated with mitotic and meiotic cell division andwhich is characterized by having a kinase domain in its N-terminus andthree PEST regions in the C-terminus, or an oligonucleotide fragment ofthe sequence which is unique to the serine/threonine kinase protein.

The kinase protein of the invention is also referred to herein as Sakfor Snk/Plk-akin kinase and the gene encoding the protein is referred toherein as sak.

In an embodiment of the invention, the purified and isolated nucleicacid molecule comprises

(a) a nucleic acid sequence encoding a protein having the amino acidsequence from amino acids 5 to 277 as shown in SEQ ID NO:2; (b) nucleicacid sequences complementary to (a); (c) nucleic acid sequences encodinga protein having at least 43% identity to (a); or, (d) a fragment of (a)or (b) that is at least 15 bases and which will hybridize to (a) or (b)under stringent hybridization conditions.

Preferably, the purified and isolated nucleic acid molecule comprises

(a) a nucleic acid Sequence corresponding to nucleic acids 218 to 1036as shown in SEQ ID NO:1, wherein T can also be U;

(b) nucleic acid sequences complementary to (a);

(c) nucleic acid sequences which are at least 56% identical to (a);

(d) a fragment of (a) or (b) that is at least 15 bases and which willhybridize to (a) or (b) under stringent hybridization conditions.

In another embodiment of the invention, the purified and isolatednucleic acid molecule comprises

(a) a nucleic acid sequence encoding a protein having the amino acidsequence as shown in SEQ ID NO:2;

(b) nucleic acid sequences complementary to (a);

(c) nucleic acid sequences encoding a protein having at least 43%identity to (a); or,

(d) a fragment of (a) or (b) that is at least 15 bases and which willhybridize to (a) or (b) under stringent hybridization conditions.

Most preferably, the purified and isolated nucleic acid moleculecomprises

(a) a nucleic acid sequence as shown in SEQ ID NO:1, wherein T can alsobe U;

(b) nucleic acid sequences complementary to (a);

(c) nucleic acid sequences which are at least 56% identical to (a); or,

(d) a fragment of (a) or (b) that is at least 15 bases and which willhybridize to (a) or (b) under stringent hybridization conditions.

In a further embodiment of the invention, the purified and isolatednucleic acid molecule comprises (a) a nucleic acid sequence encoding aprotein having the amino acid sequence as shown in SEQ ID NO:4; (b)nucleic acid sequences complementary to (a); (c) nucleic acid sequencesencoding proteins having at least 43% identity with amino acids 5 to 277in SEQ ID NO:4; or, (d) a fragment of (a) or (b) that is at least 15bases and which will hybridize to (a) or (b) under stringenthybridization conditions. Preferably, the purified and isolated nucleicacid molecule comprises (a) a nucleic acid sequence as shown in SEQ IDNO:3, wherein T can also be U; (b) nucleic acid sequences complementaryto (a); (c) nucleic acid sequences having at least 56% identity tonucleotides 218 to 1036 in SEQ ID NO: 3; or, (d) a fragment of (a) or(b) that is at least 15 bases and which will hybridize to (a) or (b)under stringent hybridization conditions.

In a still further embodiment of the invention, the purified andisolated nucleic acid molecule comprises (a) a nucleic acid sequenceencoding a protein having the amino acid sequence as shown in SEQ IDNO:6; (b) nucleic acid sequences complementary to (a); (c) nucleic acidsequences encoding a protein having at least 43% identity with aminoacids 218 to 1036 in SEQ. ID. NO:6; or, (d) a fragment of (a) or (b)that is at least 15 bases and which will hybridize to (a) or (b) understringent hybridization conditions. Preferably, the purified andisolated nucleic acid molecule comprises (a) a nucleic acid sequence asshown in SEQ ID NO:5, wherein T can also be U; (b) nucleic acidsequences complementary to (a); (c) nucleic acid sequences having atleast 56% identity with nucleotides 218 to 1036 of SEQ ID NO:5; or, (d)a fragment of (a) or (b) that is at least 15 bases and which willhybridize to (a) or (b) under stringent hybridization conditions.

It is contemplated that a nucleic acid molecule of the invention may beprepared having deletion and insertion mutations. For example, thekinase domain or parts thereof may be deleted.

The invention further contemplates a purified and isolated doublestranded nucleic acid molecule containing a nucleic acid molecule of theinvention or a fragment thereof, hydrogen bonded to a complementarynucleic acid base sequence.

The nucleic acid molecules of the invention, or fragments thereof may beinserted into an appropriate expression vector, i.e. a vector whichcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence. Accordingly, recombinant DNAmolecules adapted for transformation of a host cell may be constructedwhich comprise a nucleic acid molecule of the invention and one or moretranscription and translation elements operatively linked to the nucleicacid molecule.

In an embodiment of the invention, a recombinant molecule is providedwhich contains a nucleic acid molecule of the invention having adeletion or insertion mutation. Such a recombinant molecule may furthercomprise a reporter gene.

The recombinant molecule can be used to prepare transformed host cellsexpressing the protein or part thereof encoded by a nucleic acidmolecule of the invention. Therefore, the invention further provideshost cells containing a recombinant molecule of the invention. Theinvention also contemplates transgenic non-human mammals whose germcells and somatic cells contain a recombinant molecule of the invention.

The invention further provides a method for preparing a novelserine/threonine kinase protein or isoforms or parts thereof utilizingthe purified and isolated nucleic acid molecules of the invention.

The invention further broadly contemplates a purified and isolatedserine/threonine kinase protein which is associated with mitotic andmeiotic cell division and which is characterized by having a kinasedomain in its N-terminus and three PEST regions in the C-terminus, or anisoform, or part of the protein. In a preferred embodiment, a purifiedserine/threonine kinase protein is provided which has the amino acidsequence as shown in SEQ ID NO:2, SEQ. ID. NO:4 or SEQ. ID. NO:6, or asequence having at least 43% identity with amino acids 5 to 277 in SEQID NO:2, SEQ. ID. NO:4 or SEQ. ID. NO:6. The serine/threonine kinaseprotein of the invention may also be catalytically activated throughbinding to positive and/or negative regulators.

The kinase protein of the invention, or isoforms or parts thereof, maybe conjugated with other molecules, such as proteins or polypeptides.This may be accomplished, for example, by the synthesis of N-terminal orC-terminal fusion proteins. In a preferred embodiment a fusion proteinis provided comprising a part of the protein of the invention,preferably the kinase domain, or sequences having at least 43% identitythereto.

The invention also permits the construction of nucleotide probes whichare unique to the nucleic acid molecules of the invention andaccordingly to the novel serine/threonine kinase protein of theinvention or an isoform, or part of the protein. Thus, the inventionalso relates to a probe comprising a nucleotide sequence coding for aprotein which displays the properties of the novel serine threoninekinase of the invention or a part which is unique to the protein. Theprobe may be labelled, for example, with a detectable substance and itmay be used to select from a mixture of nucleotide sequences anucleotide sequence coding for a protein which displays the propertiesof the novel serine/threonine kinase protein of the invention, or a partthereof.

The invention further provides a method for identifying a substancewhich is capable of binding to the novel serine/threonine kinase proteinof the invention, or an isoform or part of the protein, comprisingreacting the novel serine/threonine kinase protein of the invention, orisoform or part of the protein, with at least one substance whichpotentially can bind with the kinase protein, or isoform, or part of theprotein, under conditions which permit the formation of substance-kinaseprotein complexes, and assaying for substance-kinase protein complexes,for free substance, for non-complexed kinase proteins, or for activationof the kinase proteins.

In an embodiment of the method, positive and/or negative regulators ofthe serine/threonine kinase protein of the invention are identifiedwhich are capable of binding to and activating the novelserine/threonine kinase protein of the invention. Regulators which bindto and activate the novel serine/threonine kinase protein of theinvention may be identified by assaying for serine/threonine kinaseactivity.

The invention still further provides a method for identifying asubstance which is a substrate of the novel serine/threonine kinaseprotein of the invention, or an isoform or part of the protein,comprising reacting an activated serine/threonine kinase protein of theinvention, or part of the protein, preferably the kinase domain, with atleast one substance which potentially is a substrate of the kinaseprotein, or part of the protein, under conditions which permit thephosphorylation of serine/threonine residues, and assaying forphosphorylation of the substance.

Still further, the invention provides a method for assaying a medium forthe presence of an agonist or antagonist of the interaction of aserine/threonine kinase protein of the invention or an isoform or partthereof, and a substance which binds to the serine/threonine kinaseprotein and activates the kinase protein, or isoform or part thereof, ora substance which is a substrate of the kinase protein. In anembodiment, the method comprises providing a known concentration of aserine/threonine kinase protein of the invention, or an isoform or partof the protein, incubating the kinase protein, isoform or part of theprotein with a substance which is a substrate of the kinase protein, orisoform or part of the protein, and a suspected agonist or antagonistsubstance, under conditions which permit the phosphorylation of thesubstrate, and assaying for phosphorylation of the substrate. In asecond embodiment, the method comprises providing a known concentrationof a serine/threonine kinase protein of the invention, or an isoform orpart of the protein, incubating the kinase protein with a substancewhich is capable of binding to and activating the kinase protein, orisoform or part of the protein, and a suspected agonist or antagonistsubstance under conditions which permit the formation ofsubstance-protein complexes, and assaying for activation of the kinaseprotein. The methods of the invention permit the identification ofpotential stimulators or inhibitors of cell proliferation which will beuseful in the treatment of proliferative disorders.

The invention further contemplates antibodies having specificity againstan epitope of the kinase protein of the invention or an isoform or partof the protein which is unique to the kinase protein. Antibodies may belabelled with a detectable substance and they may be used to detect thenovel kinase protein of the invention in tissues and cells. Theantibodies may accordingly be used to monitor cell proliferation.

Substances which affect cell proliferation may be identified using themethods of the invention by comparing the pattern and level ofexpression of the novel kinase protein of the invention in tissues andcells in the presence and in the absence of the substance. Thus, theinvention provides a method for screening for substances havingpharmaceutical utility in the treatment and diagnosis of proliferativedisorders.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the drawingsin which:

FIG. 1 is a schematic diagram of the sak cDNAs and nucleotide sequence;

FIG. 2 shows the nucleotide sequence of the sak-a transcriptional unitand the deduced amino acid sequence (SEQ ID NOS3 and 4);

FIG. 3 shows the nucleotide and predicted amino acid sequences of thecoding region of sak-b, beginning from the AG dinucleotide that marksthe point of sequence divergence from sak-a (SEQ ID NOS:5 and 6);

FIG. 4 shows an amino acid alignment of the kinase domains of Sak,Drosophila polo, murine plk, murine snk and yeast CDC5 (SEQ ID NOS:10 to14);

FIG. 5 is a gel showing Northern analysis of sak-a expression in murinetissues;

FIG. 6A shows a bright field photomicrograph of sagittal sectionsthrough 7.5 dpc embryo embedded in the uterus hybridized with a sak-aprobe;

FIG. 6B shows a dark field photomicrograph of sagittal sections through7.5 dpc embryo embedded in the uterus hybridized with a sak-a probe;

FIG. 6C shows a bright field photomicrograph of sagittal sectionsthrough a 11.5 dpc embryo hybridized with a sak-a probe;

FIG. 6D shows a dark field photomicrograph of sagittal sections througha 11.5 dpc embryo hybridized with a sak-a probe;

FIG. 6E shows a bright field photomicrograph of sagittal sectionsthrough a 17.5 dpc embryo hybridized with a sak-a probe;

FIG. 6F shows a dark field photomicrograph of sagittal sections througha 17.5 dpc embryo hybridized with a sak-a probe;

FIG. 6G shows a bright field photomicrograph of a coronal sectionthrough a 13.5 dpc embryo;

FIG. 6H shows a bright field photomicrograph of a coronal sectionthrough a 13.5 dpc embryo with adjacent sections probed with sak-a;

FIG. 6I shows a bright field photomicrograph of a coronal sectionthrough a 13.5 dpc embryo with adjacent sections probed with SCG10;

FIG. 6J shows a bright field photomicrograph of a coronal sectionthrough a 13.5 dpc embryo with adjacent sections probed with sense sak-acontrol;

FIG. 6K shows a bright field photomicrograph of sagittal sectionsthrough a 17.5 dpc embryo hybridized with a sak-a probe;

FIG. 6L shows a dark field photomicrograph of sagittal sections througha 17.5 dpc embryo hybridized with a sak-a probe;

FIG. 6M shows a bright field photomicrograph of sagittal sectionsthrough a 17.5 dpc embryo hybridized with a sak-a probe;

FIG. 6N shows a dark field photomicrograph of sagittal sections througha 17.5 dpc embryo hybridized with a sak-a probe;

FIG. 7A shows a bright field photomicrograph of sections through theovary of a superovulated female induced by pregnant mare serumgonadotropins;

FIG. 7B shows a dark field photomicrograph of sections through the ovaryof a superovulated female induced by pregnant mare serum gonadotropins;

FIG. 7C shows a bright field photomicrograph of sections through anadult testis;

FIG. 7D shows a dark field photomicrograph of sections through an adulttestis;

FIG. 7E shows a bright field photomicrograph of sections through theadult gut; and

FIG. 7F shows a dark field photomicrograph of sections through the adultgut.

DETAILED DESCRIPTION OF THE INVENTION I. Characterization of NucleicAcid Molecules and Proteins of the Invention

As hereinbefore mentioned, the present inventors have identified andsequenced nucleic acid molecules encoding a novel serine/threoninekinase protein. The sak-a and sak-b cDNA appear to representalternatively spliced forms of the gene given that the sequences divergeafter an AG dinucleotide (nt 1456, 1457), a sequence frequently found 5'to splice-donor sites. FIG. 1 shows the open reading frames (blackboxes) of the Sak-a and Sak-b transcriptional units which are flanked byATG start and TAA stop codons. In FIG. 1, the point of sequencedivergence is marked in Sak-b by the striped box. The sak-atranscriptional unit encodes a 925 amino acid protein (M_(r) =103 kDa)and the sak-b encoded protein is 464 amino acids in length (M_(r)=53kDa), with the first 416 amino acids of both proteins being identicalbut each having different C-terminal tails of 509 (FIG. 2, SEQ ID NO:3)and 48 amino acids (FIG. 3, SEQ ID NO:5), respectively. The N-terminalregion common to both sak-a and sak-b proteins has significant homologyto the polo subfamily of serine/threonine kinases and they have a commonstructural organization with the kinase domain located in theN-terminus, followed by a C-terminal domain. The sak kinase domain alsodiverges significantly from other members of the polo subfamily insubdomain VI-B. Within this subdomain, the threonine residue in the saksequence DLTLSN (SEQ ID NO:7) is in a position normally occupied by alysine, not only in proteins within the polo subfamily, but in themajority of serine/threonine kinases. The serine residue in thissequence is replaced by a glycine in all other members of the polosubfamily. Sak also has the sequence GTPNYISPE (SEQ ID NO:8) insubdomain VIII which matches closely with the GTXXYXAPE (SEQ ID NO:9)consensus sequence characteristic of serine/threonine kinases (Hanks, S.K. & Quinn, A. M. (1991) Methods. Enzymol. 200, 38-63). A 30 amino acidhomology domain identified in the C-terminus of the polo, CDC5, plk andsnk kinases is absent in both of the predicted sak proteins (Clay, F.J., McEwen, S. J., Bertoncello, I., Wilks, A. F. & Dunn, A. R. (1993)Proc. Natl. Acad. Sci. USA 90, 4882-4886). Three PEST regions, eachcontaining 2 sequences in tandem in Sak-a and one in Sak-b, wereidentified in the C-terminal region of the proteins. PEST sequences arerich in proline, serine, threonine, aspartate and glutamate residues,and flanked on either side by a basic amino acid.

In accordance with an embodiment of the invention a purified andisolated nucleic acid molecule is provided containing a sequenceencoding a protein having the amino acid sequence as shown in SEQ IDNO:2. Most preferably, the purified and isolated nucleic acid moleculeof the invention contains a nucleic acid sequence as shown in SEQ IDNO:1. The nucleic acid and amino acid sequences in SEQ. ID. NO:1 and 2,respectively, represent the nucleic acid and amino acid sequence of theN-terminal region of the Sak-a and Sak-b isoforms.

In accordance with another embodiment of the invention a purified andisolated nucleic acid molecule is provided containing a sequenceencoding a protein having the amino acid sequence as shown in SEQ IDNO:4. Most preferably, the purified and isolated nucleic acid moleculeof the invention contains a nucleic acid sequence as shown in SEQ IDNO:3. The nucleic acid and amino acid sequences in SEQ. ID. NO:3 and 4,respectively, represent the nucleic acid and amino acid sequences of theSak-a isoform of the serine/threonine kinase protein of the invention.

In accordance with still another embodiment of the invention a purifiedand isolated nucleic acid molecule is provided containing a sequenceencoding a protein having the amino acid sequence as shown in SEQ IDNO:6. Most preferably, the purified and isolated nucleic acid moleculeof the invention contains a nucleic acid sequence as shown in SEQ IDNO:5. The nucleic acid and amino acid sequences in SEQ. ID. NO:5 and 6,respectively, represent the nucleic acid and amino acid sequences of theSak-b isoform of the serine/threonine kinase protein of the invention.

Fragments of the nucleic acid molecules are contemplated by the presentinvention. In a preferred embodiment, the fragments include fragments ofthe nucleotide sequences as shown in SEQ. ID. NO:1, NO:3, and NO:5 andthat have at least 15 bases, and which are capable of hybridizing to thenucleotide sequence as shown in SEQ ID NO. 1, NO.3, and NO. 5respectively under stringent hybridization conditions as describedherein. These fragments may encode, for example, the kinase domain(amino acids 5 to 277 shown in SEQ ID NO:2, 4 and 6) or the carboxy tail(amino acids 417 to 926 in SEQ ID NO:4, and amino acids 417 to 465 inSEQ ID. NO. 6).

It will also be appreciated that a double stranded nucleotide sequencecomprising a nucleic acid molecule of the invention or a fragmentthereof, hydrogen bonded to a complementary nucleotide base sequence,and an RNA made by transcription of this double stranded nucleotidesequence are contemplated by the present invention.

Further, it will be appreciated that the invention includes nucleic acidor amino acid sequences which have substantial sequence identity withthe nucleic acid and amino acid sequences shown in SEQ ID NOS:1 to 6 andin FIGS. 2 and 3, and fragments thereof. The term "sequences havingsubstantial sequence identity" means those nucleic acid and amino acidsequences which have slight or inconsequential sequence variations fromthe sequences disclosed in FIGS. 2 and 3 and in SEQ ID NOS: 1 to 6, i.e.the sequences function in substantially the same manner to producesubstantially the same polypeptides as the actual sequences. Thevariations may be attributable to local mutations or structuralmodifications.

Nucleic acid sequences having substantial identity include nucleic acidsequences which encode proteins having at least 43% sequence identitywith the amino acid sequence corresponding to amino acids 5 to 277 asshown in SEQ. ID. NO:2; nucleic acid sequences having at least 56%identity with the nucleic acid sequence corresponding to nucleic acids218 to 1036 as shown in SEQ. ID. NO.:1; and fragments thereof having atleast 15 bases which will hybridize to these sequences under stringenthybridization conditions.

Stringent hybridization conditions are those which are stringent enoughto provide specificity, reduce the number of mismatches and yet aresufficiently flexible to allow formation of stable hybrids at anacceptable rate. Such conditions are known to those skilled in the artand are described, for example, in Sambrook, et al, (1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor). By way of exampleonly, stringent hybridization with short nucleotides may be carried outat 5°-10° below the T_(m) using high concentrations of probe such as0.01-1.0 pmole/ml.

The invention further provides amino acid sequences which havesubstantial identity with the amino acid sequences shown in SEQ ID NO:2,NO.:4 and NO.:6 and in FIGS. 2 and 3. Substantially identical amino acidsequences include sequences having at least 43% sequence identity in thekinase domain set out in SEQ ID NOS:2, 4 and 6 (amino acids 5 to 277).The invention still further provides peptides which are unique to theserine/threonine kinase protein of the invention. Preferably, thepeptides have at least 10 to 20 amino acids. For example the peptidesmay contain the amino acid sequences of the kinase domain, or subdomainsthereof such as subdomain VI-B shown in FIG. 4, or the amino acidsequences of the C-terminal domains of Sak-a and Sak-b.

The nucleic acid sequence contained in the nucleic acid molecules of theinvention or a fragment thereof, may be inverted relative to theirnormal presentation for transcription to produce antisense nucleic acidmolecules. The antisense nucleic acid molecules may be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. The antisense nucleic acid molecules of the inventionor a fragment of the antisense sequence, preferably containing at least15 bases, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with sak mRNA or the Sak gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sak sequences areproduced under the control of a high efficiency regulatory region, theactivity of which may be determined by the cell type into which thevector is introduced.

A number of unique restriction sequences for restriction enzymes areincorporated in the nucleic acid sequences identified in SEQ ID NO:1,NO:3 and NO:5 and these provide access to nucleic acid sequences whichcode for polypeptides unique to the kinase protein of the invention.Nucleic acid sequences unique to the kinase protein of the invention orisoforms or parts thereof, can also be constructed by chemical synthesisand enzymatic ligation reactions carried out by procedures known in theart.

The invention also contemplates forms of the kinase protein whichcontain the same number and kinds of amino acids as the protein of theinvention, but having a different molecular structure and the samefunctional properties as the novel kinase protein of the invention asdescribed herein.

The present invention also includes fusion proteins containing thekinase protein of the invention, or an isoform, or part thereof. Forexample, the kinase protein or portions thereof may be conjugated with aselected protein or marker protein to produce fusion proteins.

The present invention also includes a kinase protein of the invention,or an isoform, or part thereof, preferably the kinase domain, which isenzymatically active. The catalytically active form of the protein orisoform or part thereof is also referred to herein as an "activatedserine/threonine kinase protein or isoform or part thereof" or"catalytically activated serine/threonine kinase protein or isoform orpart thereof".

II. Expression Pattern of the Kinase Protein of the Invention

A detailed study of sak expression by Northern and RNA in situ analysesshows that sak expression correlates with mitotic and meiotic activityin embryonic and adult tissues. For example, sak transcripts in theembryonic CNS are confined to the ventricular zones where neuroblastsare actively dividing and are not detected in the intermediate ormarginal zones where the post-mitotic neurons are located. In adultmice, sak is expressed in tissues with a mitotic component, includinghematopoietic tissues, the stem cells of the intestinal crypt, thegranulosa cells of developing ovarian follicles and in the matrix, orgrowth region of the hair follicle. RNA in situ analysis revealed highlevels of sak transcripts in meiotic spermatocytes and oocytes.

In addition to mitotically dividing cells, the respiratory and olfactorymucosa represent major sites of sak expression during embryogenesis. sakis expressed in the basal region of the olfactory epithelium, consistingmainly of progenitor stem cells for the olfactory neuronal lineage. Inthe respiratory system, high levels of sak expression are seen in themucous-secreting and ciliated cells epithelium of the nasal cavity andlarger airways, with levels decreasing as the respiratory tractbranches. A common feature of ciliated and dividing cells is the motilenature of the microtubular system. Defects in the organization of themicrotubular system during cell division are seen in polo¹ homozygotesand CDC5^(ts) mutants, suggesting that this family of kinases includingsak, may regulate microtubule-mediated processes.

III. Preparation of Nucleic Acid Molecules and Proteins of the Invention

The nucleic acid molecules of the invention encoding the novel kinaseprotein, or fragments thereof, may be isolated and sequenced usingprocedures known in the art. For example, the Sak coding region may becloned by transfecting a murine lymphoid cDNA library into CHOP cells,treating with the sialic acid binding lectin wheat germ agglutinin(WGA), and isolating WGA resistant CHOP clones containing a 1.5 kb cDNAinsert (17S clone, FIG. 1). The 17S partial cDNA fragment, a more 5'fragment (probe 2, FIG. 1) or a probe with sequences unique to the 3untranslated region of sak-b (probe 3,FIG. 1) may be used to obtainfull-length clones from the lymphoid cDNA library. Oligonucleotidesspecific for the novel serine/threonine kinase protein and which can beused as probes may also be identified by comparing the nucleic acidsequence of the nucleic acid molecules of the invention to knowsequences, for example, sequences of the other members of the polosubfamily. Nucleic acid molecules of the present invention encoding thenovel kinase protein and oligonucleotide fragments thereof, may also beconstructed by chemical synthesis and enzymatic ligation reactions usingprocedures known in the art.

The novel kinase protein of the invention, or isoforms or parts thereof,may be prepared using recombinant DNA methods. Accordingly, the nucleicacid molecules of the present invention having a sequence which codesfor the kinase protein of the invention, or a fragment thereof may beincorporated in a known manner into an appropriate expression vectorwhich ensures good expression of the kinase protein or isoform or partthereof. Possible expression vectors include but are not limited tocosmids, plasmids, or modified viruses, so long as the vector iscompatible with the host cell used.

The invention therefore contemplates a recombinant molecule of theinvention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary elements for the transcription andtranslation of the inserted sequence. Suitable transcription andtranslation elements may be derived from a variety of sources, includingbacterial, fungal, viral, mammalian, or insect genes. Selection ofappropriate transcription and translation elements is dependent on thehost cell chosen as discussed below, and may be readily accomplished byone of ordinary skill in the art. Examples of such elements include: atranscriptional promoter and enhancer or RNA polymerase bindingsequence, a ribosomal binding sequence, including a translationinitiation signal. Additionally, depending on the host cell chosen andthe vector employed, other genetic elements, such as an origin ofreplication, additional DNA restriction sites, enhancers, and sequencesconferring inducibility of transcription may be incorporated into theexpression vector. It will also be appreciated that the necessarytranscription and translation elements may be supplied by the nativekinase protein and/or its flanking regions.

The recombinant molecules of the invention may also contain a reportergene which facilitates the selection of host cells transformed ortransfected with a recombinant molecule of the invention. Examples ofreporter genes are genes encoding a protein such as β-galactosidase,chloramphenicol acetyltransferase, firefly luciferase, or animmunoglobulin or portion thereof such as the Fc portion of animmunoglobulin preferably IgG. In a preferred embodiment, the reportergene is lac Z. Transcription of the reporter gene is monitored bychanges in the concentration of the reporter protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. This makes it possible to visualize and assay for expressionof recombinant molecules of the invention and in particular to determinethe effect of a mutation on expression and phenotype.

Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation etc. Methodsfor transforming transfecting, etc. host cells to express foreign DNAare well known in the art (see, e.g., Itakura et al., U.S. Pat. No.4,704,362; Hinnen et al., PNAS USA 75:1929-1933, 1978; Murray et al.,U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No. 4,935,349; Hagenet al., U.S. Pat. No. 4,784,950; Axel et al., U.S. Pat. No. 4,399,216;Goeddel et al., U.S. Pat. No. 4,766,075; and Sambrook et al. MolecularCloning A Laboratory Manual, 2nd edition, Cold Spring Harbor LaboratoryPress, 1989, all of which are incorporated herein by reference and seethe detailed discussion below).

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells, including bacterial, mammalian, yeast or other fungi, viral,plant, or insect cells.

More particularly, bacterial host cells suitable for carrying out thepresent invention include E. coli, B. subtilis, Salmonella typhimurium,and various species within the genus' Pseudomonas, Streptomyces, andStaphylococcus, as well as many other bacterial species well known toone of ordinary skill in the art. Representative examples of bacterialhost cells include DH5α (Stratagene, LaJolla, Calif.), JM109 ATCC No.53323, HB101 ATCC No. 33694, and MN294. Suitable bacterial expressionvectors preferably comprise a promoter which functions in the host cell,one or more selectable phenotypic markers, and a bacterial origin ofreplication. Representative promoters include the β-lactamase(penicillinase) and lactose promoter system (see Chang et al., Nature275:615, 1978), the trp promoter (Nichols and Yanofsky, Meth inEnzymology 101:155, 1983) and the tac promoter (Russell et al., Gene 20:231, 1982). Representative selectable markers include various antibioticresistance markers such as the kanamycin or ampicillin resistance genes.Suitable expression vectors include but are not limited tobacteriophages such as lambda derivatives or plasmids such as pBR322(see Bolivar et al., Gene 2:9S, 1977), the pUC plasmids pUC18, pUC19,pUC118, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983 andVieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNH18a,pCDM8, and Bluescript M13 (Stratagene, La Jolla, Calif.).

Yeast and fungi host cells suitable for carrying out the presentinvention include, among others Saccharomyces cerevisiae, the generaPichia or Kluyveromyces and various species of the genus Aspergillus.Suitable expression vectors for yeast and fungi include, among others,YC_(p) 50 (ATCC No. 37419 ) for yeast, and the amdS cloning vector pV3(Turnbull, Bio/Technology 7:169, 1989). Protocols for the transformationof yeast are also well known to those of ordinary skill in the art. Forexample, transformation may be readily accomplished either bypreparation of spheroplasts of yeast with DNA (see Hinnen et al., PNASUSA 75:1929, 1978) or by treatment with alkaline salts such as LiCl (seeItoh et al., J. Bacteriology 153:163, 1983). Transformation of fungi mayalso be carried out using polyethylene glycol as described by Cullen etal. (Bio/Technology 5:369, 1987).

Mammalian cells suitable for carrying out the present invention include,among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No.CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCCNo. 1573), CHOP, and NS-1 cells. Suitable expression vectors fordirecting expression in mammalian cells generally include a promoter, aswell as other transcription and translation control sequences. Commonpromoters include SV40, MMTV, metallothionein-1, adenovirus Ela, CMV,immediate early, immunoglobulin heavy chain promoter and enhancer, andRSV-LTR. Protocols for the transfection of mammalian cells are wellknown to those of ordinary skill in the art. Representative methodsinclude calcium phosphate mediated electroporation, retroviral, andprotoplast fusion-mediated transfection (see Sambrook et al., supra).

Given the teachings provided herein, promoters, terminators, and methodsfor introducing expression vectors of an appropriate type into plant,avian, and insect cells may also be readily accomplished. For example,within one embodiment, Sak or derivatives thereof may be expressed fromplant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58, 1987,which reviews the use of Agrobacterium rhizogenes vectors; see alsoZambryski et al., Genetic Engineering, Principles and Methods,Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum Press, NewYork, 1984, which describes the use of expression vectors for plantcells, including, among others, pAS2022, pAS2023, and pAS2034).

Insect cells suitable for carrying out the present invention includecells and cell lines from Bombyx or Spodotera species. Suitableexpression vectors for directing expression in insect cells includeBaculoviruses such as the Autographa california nuclear polyhedrosis,virus (Miller et al. 1987, in Genetic Engineering, Vol. 8 ed. Setler, J.K. et al., Plenum Press, New York) and the Bombyx mori nuclearpolyhedrosis virus (Maeda et al., 1985, Nature 315:592).

Alternatively, the kinase protein of the invention, isoforms or partsthereof may be expressed in non-human transgenic animals such as, rats,rabbits, sheep and pigs (see Hammer et al. (Nature 315:680-683, 1985),Palmiter et al. (Science 222:809-814, 1983), Brinster et al. (Proc Natl.Acad. Sci USA 82:44384442, 1985), Palmiter and Brinster (Cell.41:343-345, 1985) and U.S. Pat. No. 4,736,866).

The kinase protein of the invention or isoforms or parts thereof mayalso be prepared by chemical synthesis using techniques well known inthe chemistry of proteins such as solid phase synthesis (Merrifield,1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenoussolution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch,Vol. 15 I and II, Thieme, Stuttgart).

The kinase protein of the invention, or isoforms or parts thereof, maybe conjugated with other molecules, such as proteins or polypeptides.This may be accomplished, for example, by the synthesis of N-terminal orC-terminal fusion proteins. Thus, fusion proteins may be prepared byfusing, through recombinant techniques, the N-terminal or C-terminal ofthe kinase protein or parts thereof, and the sequence of a selectedprotein or marker protein with a desired biological function. Theresultant fusion proteins contain the kinase protein or a portionthereof fused to the selected protein or marker protein as describedherein. Examples of proteins which may be used to prepare fusionproteins include immunoglobulins and parts thereof such as the constantregion of immunglobulin γl, and lymphokines such as gamma interferon,tumor necrosis factor, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, GM-CSF, CSF-1, G-CSF, hemaglutinin 1, and enzymessuch as β-galactosidase, chloramphenicol acetyltransferase, fireflyluciferase.

Sequences which encode the above-described proteins may generally beobtained from a variety of sources, including for example, depositorieswhich contain plasmids encoding sequences including the American TypeCulture Collection (ATCC, Rockville, Md.), and the British BiotechnologyLimited (Cowley, Oxford England). Examples of such plasmids include BBG12 (containing the GM-CSF gene coding for the mature protein of 127amino acids), BBG 6 (which contains sequences encoding gammainterferon), ATCC No. 39656 (which contains sequences encoding TNF),ATCC No. 20663 (which contains sequences encoding alpha interferon,)ATCC Nos. 31902 and 39517 (which contains sequences encoding betainterferon), ATCC No. 67024 (which contains a sequence which encodesInterleukin-1β), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (whichcontains sequences encoding Interleukin-2), ATCC Nos. 59399, 59398, and67326 (which contain sequences encoding Interleukin-3), ATCC Nos. 57592(which contains sequences encoding Interleukin-4). ATCC Nos. 59394 and59395 (which contain sequences encoding Interleukin-5), and ATCC No.67153 (which contains sequences encoding Interleukin-6.

Activated serine/threonine kinase proteins of the invention, or isoformsor parts thereof, may be prepared by reacting a serine/threonine kinaseprotein of the invention or a part thereof with a positive and/ornegative regulator which is identified using the methods of theinvention described herein.

IV. Utility of the Nucleic Acid Molecules and Proteins of the Invention

The nucleic acid molecules of the invention or fragments thereof, allowthose skilled in the art to construct nucleotide probes for use in thedetection of nucleotide sequences in biological materials. Examples ofprobes include the 17S partial cDNA fragment, a more 5' fragment (probe2, FIG. 1) and a probe with sequences unique to the 3 untranslatedregion of sak-b (probe 3, FIG. 1). A nucleotide probe may be labelledwith a detectable substance such as a radioactive label which providesfor an adequate signal and has sufficient half-life such as ³² P, ³ H,¹⁴ C or the like. Other detectable substances which may be used includeantigens that are recognized by a specific labelled antibody,fluorescent compounds, enzymelac Zs, antibodies specific for a labelledantigen, and chemiluminescense. An appropriate label may be selectedhaving regard to the rate of hybridization and binding of the probe tothe nucleotide to be detected and the amount of nucleotide available forhybridization. Labelled probes may be hybridized to nucleic acids onsolid supports such as nitrocellulose filters or nylon membranes asgenerally described in

Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).The nucleotide probes may be used to detect genes, preferably in humancells, that encode proteins related to or analogous to the kinaseprotein of the invention. The nucleotide probes may therefore be usefulin the diagnosis of proliferative disorders arising from mutations oralterations to the Sak gene or a homologue thereof.

Antisense nucleic acid molecules of the invention may be used in genetherapy to treat or prevent proliferative disorders. For a discussion ofthe regulation of gene expression using anti-sense genes see Weintraub,H. et al., Antisense RNA as a molecular tool for genetic analysis,Reviews--Trends in Genetics, Vol. 1(1) 1986. Recombinant moleculescomprising an antisense sequence or oligonucleotide fragment thereof,may be directly introduced into cells of tissues in vivo using deliveryvehicles such as retroviral vectors, adenoviral vectors and DNA virusvectors. They may also be introduced into cells in vivo using physicaltechniques such as microinjection and electroporation or chemicalmethods such as coprecipitation and incorporation of DNA into liposomes.Recombinant molecules may also be delivered in the form of an aerosol orby lavage.

The kinase protein of the invention and isoforms and parts thereof, forexample amino acids of the carboxy terminal tail, may be used to prepareantibodies. Antibodies having specificity for Sak protein may also beraised from fusion proteins created by expressing Sak fusion proteins inbacteria as described above.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies, polyclonal antibodies, antibodyfragments (e.g., Fab, and F(ab')₂ and recombinantly produced bindingpartners. Antibodies are understood to be reactive against Sak proteinif they bind with a K_(a) of greater than or equal to 10⁻⁷ M. As will beappreciated by one of ordinary skill in the art, antibodies may bedeveloped which not only bind to Sak protein, but which bind to aregulator of Sak protein, and which also block the biological activityof Sak protein. Such antibodies will be useful in the diagnosis andtreatment of proliferative disorders.

Polyclonal antibodies may be readily generated by one of ordinary skillin the art from a variety of warm-blooded animals such as horses, cows,various fowl, rabbits, mice, or rats. Briefly, Sak protein is utilizedto immunize the animal through intraperitoneal, intramuscular,intraocular, or subcutaneous injections, in conjunction with an adjuvantsuch as Freund's complete or incomplete adjuvant. Following severalbooster immunizations, samples of serum are collected and tested forreactivity to Sak protein. Particularly preferred polyclonal antiserawill give a signal on one of these assays that is at least three timesgreater than background. Once the titer of the animal has reached aplateau in terms of its reactivity to Sak protein, larger quantities ofantisera may be readily obtained either by weekly bleedings, or byexsanguinating the animal.

Monoclonal antibodies may also be readily generated using conventionaltechniques (see U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and4,411,993 which are incorporated herein by reference; see alsoMonoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, andAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988, which are also incorporated herein byreference).

Briefly, within one embodiment a subject animal such as a rat or mouseis injected with Sak protein. The Sak protein may be admixed with anadjuvant such as Freund's complete or incomplete adjuvant in order toincrease the resultant immune response. Between one and three weeksafter the initial immunization the animal may be reimmunized withanother booster immunization, and tested for reactivity to Sak proteinusing assays described above. Once the animal has plateaued in itsreactivity to Sak protein, it is sacrificed, and organs which containlarge numbers of B cells such as the spleen and lymph nodes areharvested.

Cells which are obtained from the immunized animal may be immortalizedby transfection with a virus such as the Epstein bar virus (EBV) (seeGlasky and Reading, Hybridoma 8(4):377-389, 1989). Alternatively, withina preferred embodiment, the harvested spleen and/or lymph node cellsuspensions are fused with a suitable myeloma cell in order to create a"hybridoma" which secretes monoclonal antibody. Suitable myeloma linesinclude, fox example, NS-1 (ATCC No. TIB 18), and P3X63--Ag 8.653 (ATCCNo. CRL 1580).

Following the fusion, the cells may be placed into culture platescontaining a suitable medium, such as RPMI 1640, or DMEM (Dulbecco'sModified Eagles Medium) (JRH Biosciences, Lenexa, Kans.), as well asadditional ingredients, such as Fetal Bovine Serum (FBS, ie., fromHyclone, Logan, Utah, or JRH Biosciences). Additionally, the mediumshould contain a reagent which selectively allows for the growth offused spleen and myeloma cells such as HAT (hypoxanthine, aminopterin,and thymidine) (Sigma Chemical Co., St. Louis, Mo.). After about sevendays, the resulting fused cells or hybridomas may be screened in orderto determine the presence of antibodies which are reactive against Sakprotein. A wide variety of assays may be utilized to determine thepresence antibodies which are reactive against Sak protein, includingfor example Countercurrent Immuno-Electrophoresis, Radioimmunoassays,Radioimmunoprecipitations, Enzyme-Linked Immuno-Sorbent Assays (ELISA),Dot Blot assays, Inhibition or Competition Assays, and sandwich assays(see U.S. Pat. Nos. 4,376,110 and 4,186,530; see also Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, 1988). Following several clonal dilutions and reassays, ahybridoma producing antibodies reactive against Sak protein may beisolated.

Other techniques may also be utilized to construct monoclonal antibodies(see William D. Huse et al., "Generation of a Large CombinationalLibrary of the Immunoglobulin Repertoire in Phage Lambda," Science246:1275-1281, December 1989; see also L. Sastry et al., "Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library," Proc Natl. Acad. Sci USA 86:5728-5732,August 1989; see also Michelle Alting-Mees et al., "Monoclonal AntibodyExpression Libraries: A Rapid Alternative to Hybridomas," Strategies inMolecular Biology 3:1-9, January 1990; these references describe acommercial system available from Stratacyte, La Jolla, Calif., whichenables the production of antibodies through recombinant techniques).Briefly, mRNA is isolated from a B cell population, and utilized tocreate heavy and light chain immunoglobulin cDNA expression libraries inthe λImmunoZap(H) and λImmunoZap(L) vectors. These vectors may bescreened individually or co-expressed to form Fab fragments orantibodies (see Huse et al. supra; see also Sastry et al., supra).Positive plaques may subsequently be converted to a non-lytic plasmidwhich allows high level expression of monoclonal antibody fragments fromE. coli.

Similarly, binding partners may also be constructed utilizingrecombinant DNA techniques to incorporate the variable regions of a genewhich encodes a specifically binding antibody. Within one embodiment,the genes which encode the variable region from a hybridoma producing amonoclonal antibody of interest are amplified using nucleotide primersfor the variable region. These primers may be synthesized by one ofordinary skill in the art, or may be purchased from commerciallyavailable sources. Primers for mouse and human variable regionsincluding, among others, primers for V_(Ha), V_(Hb), V_(Hc), V_(Hd),C_(H1), V_(L) and C_(L) regions are available from Stratacyte (La Jolla,Calif.). These primers may be utilized to amplify heavy or light chainvariable regions, which may then be inserted into vectors such asImmunoZAP™ H or ImmunoZAP™ L (Stratacyte), respectively. These vectorsmay then be introduced into E. coli for expression. Utilizing thesetechniques, large amounts of a single-chain protein containing a fusionof the VH and VL domains may be produced (See Bird et al., Science242:423-426, 1988). In addition, such techniques may be utilized tochange a "murine" antibody to a "human" antibody, without altering thebinding specificity of the antibody.

Once suitable antibodies or binding partners have been obtained, theymay be isolated or purified by many techniques well known to those ofordinary skill in the art (see Antibodies: A Laboratory Manual, Harlowand Lane (eds.), Cold Spring Harbor Laboratory Press, 1988). Suitabletechniques include peptide or protein affinity columns, HPLC or RP-HPLC,purification on protein A or protein G columns, or any combination ofthese techniques.

The polyclonal or monoclonal antibodies may be used to detect the kinaseprotein of the invention, or isoforms or parts thereof, in variousbiological materials, for example they may be used in an Elisa,radioimmunoassay or histochemical tests. Thus, the antibodies may beused to quantify the amount of a kinase protein of the invention, or anisoform or part thereof, in a sample in order to determine its role inparticular cellular events or pathological states and to diagnose andtreat such pathological states.

In particular, the polyclonal and monoclonal antibodies of the inventionmay be used in immunohistochemical analyses, for example, at thecellular and sub-subcellular level, to detect the novel kinase proteinof the invention, to localise it to particular cells and tissues and tospecific subcellular locations, and to quantitate the level ofexpression.

Cytochemical techniques known in the art for localizing antigens usinglight and electron microscopy may be used to detect the novel kinaseprotein of the invention. Generally, an antibody of the invention may belabelled with a detectable substance and the novel kinase protein of theinvention may be localised in tissue based upon the presence of thedetectable substance. Examples of detectable substances include variousenzymes, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,biotin, alkaline phosphatase, β-galactosidase, or acetylcholinesterase;examples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include radioactive iodine I¹²⁵, I¹³¹ ortritium. Antibodies may also be coupled to electron dense substances,such as ferritin or colloidal gold, which are readily visualised byelectron microscopy.

Radioactive labelled materials may be prepared by radiolabeling with ¹²⁵I by the chloramine-T method (Greenwood et al, Biochem. J. 89:114,1963), the lactoperoxidase method (Marchalonis et al, Biochem. J.124:921, 1971), the Bolton-Hunter method (Bolton and Hunter, Biochem. J.133:529, 1973 and Bolton Review 18, Amersham International Limited,Buckinghamshire, England, 1977), the iodogen method (Fraker and Speck,Biochem. Biophys. Res. Commun. 80:849, 1978), the Iodo-beads method(Markwell Anal. Biochem. 125:427, 1982) or with tritium by reductivemethylation (Tack et al., J. Biol. Chem. 255:8842, 1980).

Known coupling methods (for example Wilson and Nakane, in"Immunofluorescence and Related Staining Techniques", W. Knapp et al,eds, p. 215, Elsevier/North-Holland, Amsterdam & New York, 1978; P.Tijssen and E. Kurstak, Anal. Biochem. 136:451, 1984) may be used toprepare enzyme labelled materials. Fluorescent labelled materials may beprepared by reacting the material with umbelliferone, fluorescein,fluorescein isothiocyanate, dichlorotriazinylamine fluorescein, dansylchloride, derivatives of rhodamine such as tetramethyl rhodamineisothiocyanate, or phycoerythrin.

Indirect methods may also be employed in which the primaryantigen-antibody reaction is amplified by the introduction of a secondantibody, having specificity for the antibody reactive against the novelkinase protein of the invention. By way of example, if the antibodyhaving specificity against the novel kinase protein of the invention isa rabbit IgG antibody, the second antibody may be goat anti-rabbitgamma-globulin labelled with a detectable substance as described herein.

Where a radioactive label is used as a detectable substance, the novelkinase protein of the invention may be localized by radioautography. Theresults of radioautography may be quantitated by determining the densityof particles in the radioautographs by various optical methods, or bycounting the grains.

As discussed above, the expression patterns found for the novel proteinkinase of the invention indicate that it plays unique and importantroles in cell proliferation. Therefore, the above described methods fordetecting nucleic acid molecules and fragments thereof and Sak proteinand parts thereof, can be used to monitor cell proliferation bydetecting and localizing the novel kinase protein of the invention inorganisms, tissues, and embryos.

It would also be apparent to one skilled in the art that the abovedescribed methods may be used to study the developmental expression ofsak and, accordingly, will provide further insight into the role of Sakprotein in cell proliferation.

The finding of a novel serine/threonine kinase protein which isexpressed in proliferating cells permits the identification ofsubstances which affect cell proliferation. A substance which affectsexpression of Sak protein and thus cell proliferation may be assayedusing the above described methods for detecting nucleic acid moleculesand fragments thereof and Sak protein or isoforms or parts thereof, bycomparing the pattern and level of expression of the Sak protein orisoforms or parts thereof, in the presence and absence of the substance.

The invention also provides methods for identifying substances which arecapable of binding to the Sak protein, or isoforms or parts thereof.Substances which can bind with the kinase protein of the invention orisoforms or parts thereof, may be identified by reacting the novelkinase protein of the invention or isoform or part of the protein, witha substance which potentially binds to the novel kinase protein, orisoform or part of the protein such as the kinase domain, and assayingfor substance-protein complexes, for free substance or for non-complexedkinase protein, or isoform, or part of the protein, or for activation ofthe kinase protein.

Conditions which permit the formation of substance-protein complexes maybe selected having regard to factors such as the nature and amounts ofthe substance and the kinase protein.

The substance-protein complex, free substance or non-complexed proteinsor isoforms or parts thereof, may be isolated by conventional isolationtechniques, for example, salting out, chromatography, electrophoresis,gel filtration, fractionation, absorption, polyacrylamide gelelectrophoresis, agglutination, or combinations thereof.

To facilitate the assay of the components, antibody against the kinaseprotein or the substance, or a labelled kinase protein, or a labelledsubstance may be utilized. Antibodies, kinase protein or substance maybe labelled with a detectable substance as described above.

The kinase protein or isoforms or parts thereof, or substance used inthe method of the invention may be insolubilized. For example, thekinase protein or substance may be bound to a suitable carrier. Examplesof suitable carriers are agarose, cellulose, dextran, Sephadex,Sepharose, carboxymethyl cellulose polystyrene, filter paper,ion-exchange resin, plastic film, plastic tube, glass beads,polyamine-methyl vinyl-ether-maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carriermay be in the shape of, for example, a tube, test plate, beads, disc,sphere etc.

The insolubilized kinase protein or substance may be prepared byreacting the material with a suitable insoluble carrier using knownchemical or physical methods, for example, cyanogen bromide coupling.

The above mentioned methods of the invention may be used to identifypositive and/or negative regulators which bind with and catalyticallyactivate the novel kinase protein of the invention thereby affectingcell proliferation. Identification and isolation of such Sak proteinregulators will permit studies of the role of the regulators in theregulation of cell proliferation and permit the development ofsubstances which affect this role, such as functional or non-functionalanalogues of the regulators. It will be appreciated that such substanceswill be useful as pharmaceuticals to modulate cell proliferation.Regulators which bind to and catalytically activate the novel kinaseprotein of the invention may be identified by assaying for proteinserine/threonine kinase activity. Serine/threonine kinase proteinactivity may be assayed using known techniques such as those usinganti-phosphoserine/phosphothreonine antibodies and labelled phosphorous.For example, immunoblots of a substance containing serine/threoninewhich is treated with a cataytically active kinase protein of theinvention, or isoform or part thereof may be analyzed by autoradiography(³² P-labelled samples) or may be blocked and probed withantiphosphoserine or phosphothreonine antibodies.

The invention further contemplates a method for identifying a substancewhich is a substrate of an activated kinase protein of the invention oran isoform or part of the activated protein, comprising reacting anactivated kinase protein of the invention, or isoform or part of theprotein, with at least one substance which potentially is a substrate ofthe kinase protein, or isoform or part of the protein, under conditionswhich permit the phosporylation of the substance, and assaying forphosphorylation of the substance.

An activated kinase protein of the invention, or isoform or part thereofmay be prepared by binding of a positive and/or negative regulator tothe kinase protein of the invention, isoform or part thereof whichresults in activation of the catalytic domain.

Conditions which permit the phosphorylation of the substance may beselected having regard to factors such as the nature and amounts of thesubstance and the kinase protein. Phosphorylation of the substance maybe determined using for example, labelled phosphorous as describedabove.

The invention also contemplates a method for assaying for an agonist orantagonist of the binding of the novel kinase protein of the invention,an isoform or part thereof, with a substance which is capable of bindingwith the novel kinase protein, preferably a positive and/or negativeregulator or a substance which is a substrate. The agonist or antagonistmay be an endogenous physiological substance or it may be a natural orsynthetic drug.

In accordance with a preferred embodiment, a method is provided whichcomprises providing a known concentration of the novel kinase protein ofthe invention or an isoform or part thereof, incubating the protein, orisoform or part thereof, with a positive and/or negative regulator whichcan bind to and catalytically activate the protein or isoform or partthereof, and optionally a substance which is a substrate, and asuspected agonist or antagonist and assaying for activation of thekinase protein, or isoform or part thereof. Methods for assaying foractivation of the kinase protein are described herein.

It will be understood that the agonists and antagonists that can beassayed using the methods of the invention may act on one or more of thebinding sites on the kinase protein or regulator, including agonistbinding sites, competitive antagonist binding sites, non-competitiveantagonist binding sites or allosteric sites.

The invention also makes it possible to screen for antagonists thatinhibit the effects of an agonist of the interaction of sak protein witha Sak protein regulator or a substrate of the Sak protein. Thus, theinvention may be used to assay for a substance that competes for thesame regulator or substrate binding site on the novel kinase protein ofthe invention.

The invention further provides a method for assaying for a substancethat affects cell proliferation comprising administering to a non-humananimal or to a tissue of an animal, a substance suspected of affectingcell proliferation, and detecting, and optionally quantitating, thenovel kinase protein of the invention in the non-human animal or tissue.

In another embodiment, the method may be used to assay for a substancethat affects cell proliferation, comprising administering a substancesuspected of affecting cell proliferation to a non-human animal having aproliferative disorder and detecting, and optionally quantitating, thenovel protein kinase of the invention in the non-human animal. Examplesof non-human animals having proliferative disorders includetransplantable tumor models such as the B16 melanoma cell model. Theeffect of a substance may be assayed by injecting B16 melanoma cellsinto mice along with anti-sense sak and the substance to determine ifthe substance effects the ability of the antisense molecule to suppressgrowth.

Substances which are capable of binding to the kinase protein of theinvention or isoforms or parts thereof, particularly regulators,agonists and antagonists of the binding of regulators and substrates ofSak protein identified by the methods of the invention, antisensenucleic acid molecules of the invention, and antibodies of the inventionmay be used for stimulating or inhibiting cell proliferation. Theregulators, agonists and antagonists, substrates etc. may accordingly beused to stimulate or inhibit cell proliferation associated withdisorders including various forms of cancer such as leukemias, lymphomas(Hodgkins and non-Hodgkins), sarcomas, melanomas, adenomas, carcinomasof solid tissue, hypoxic tumors, squamous cell carcinomas of the mouth,throat, larynx, and lung, genitourinary cancers such as cervical andbladder cancer, hematopoietic cancers, head and neck cancers, andnervous system cancers, benign lesions such as papillomas,arthrosclerosis, angiogenesis, and viral infections, in particular HIVinfections; and autoimmune diseases including systemic lupuserythematosus, Wegener's granulomatosis, rheumatoid arthritis,sarcoidosis, polyarthritis, pemphigus, pemphigoid, erythema multiforme,Sjogren's syndrome, inflammatory bowel disease, multiple sclerosis,myasthenia gravis, keratitis, scleritis, Type I diabetes,insulin-dependent diabetes mellitus, Lupus Nephritis, allergicencephalomyelitis. Substances which stimulate cell proliferationidentified using the methods of the invention may be useful in thetreatment of conditions involving damaged cells including conditions inwhich degeneration of tissue occurs such as arthropathy, boneresorption, inflammatory disease, degenerative disorders of the centralnervous system; and for promoting wound healing.

Sak transcripts were found in meiotic spermatocytes and oocytessuggesting that substances identified using the method of the invention,and the antibodies and anti-sense nucleic acid molecules describedherein may be useful as contraceptives. The substances, antibodies andanti-sense nucleic acid molecules may also affect the fertility ofmammalian sperm.

The invention also relates to a pharmaceutical composition comprising asubstance identified using the methods described herein or antibodiesdescribed herein.

The pharmaceutical compositions of the invention contain the substanceor antibodies, alone or together with other active substances. Suchpharmaceutical compositions can be for oral, topical, rectal,parenteral, local, inhalant or intracerebral use. They are therefore insolid or semisolid form, for example pills, tablets, creams, gelatincapsules, capsules, suppositories, soft gelatin capsules, gels,membranes, tubelets.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., U.S.A. 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

The utility of the substances, antibodies, antisense nucleic acidmolecules, and compositions of the invention may be confirmed in animalexperimental model systems. For example, therapeutic utility inproliferative disorders may be tested by examining the ability of asubstance etc. to suppress the growth of a transplantable tumor such asB16 melanoma cells. The utility of a substance etc. in fibroticconditions may be tested by examining the susceptibility of mice to theinduction of pulmonary fibrosis by bleomucin sulfate (Baecher-Allan,Regional Immunology 5(3-4):207, 1993). The well-characterized pig modelof radiation induced fibrosis described in Martin et al, RadiationResearch 134(1)63, 1993, and the experimental glomerulonephritis modeldescribed in Border et al, Nature 360:361, 1992 may also be utilised.Other models which may be useful in confirming the utility of thesubstances, antibodies and nucleic acid molecules of the presentinvention include those for wound healing (e.g. the fetal tissue repairmodel described in Bleacher et al. Dermatologic Clinics 11(4):677,1993), bone repair (e.g. bone induction in rats--Yasko, A. W., et al.,Orthop. Trans. 15:501, 1991; sheep femur--Gerhart T. N. et al, Trans.37Annual Meeting Orthop. Res. Soc. Anaheim, Calif. Catherson, B., ed)16(1), p.172, 1991; and dog mandible--Toriumi D. M. et al, Archiv.Otolarynogol. Head Neck Surg. 117:1101-1112, 1991), and autoimmunediseases (e.g. MRL-lpr/ipr mice are a model for systemic lupuserythematosus, and NZBxNZWf1 mice which demonstrate clinical symptomscomparable to those found with human autoimmune diseases--Theofilopoulosand Dixon, Adv. Immunol. 37, 1985).

The invention also provides methods for studying the function of the Sakprotein. Cells, tissues, and non-human animals lacking in Sak expressionor partially lacking in Sak expression may be developed usingrecombinant molecules of the invention having specific deletion orinsertion mutations in the sak gene. For example, the kinase domain orparts thereof may be deleted. A recombinant molecule may be used toinactivate or alter the endogenous gene by homologous recombination, andthereby create a Sak deficient cell, tissue or animal.

Sak alleles may be generated in cells, such as embryonic stem cells bydeletion mutation. A recombinant sak gene molecule may also beengineered to contain an insertion mutation which inactivates Sak. Forexample, a recombinant molecule may be engineered with an Asp mutationin the nucleic acids encoding the amino acid stretch KLAD*FGLAR (*markspoint of mutation) in the kinase domain (van den Heuvel and E. Harlow,1993, Science, 262:2050-2054). Such recombinant molecules may then beintroduced into a cell, such as an embryonic stem cell, by a techniquesuch as transfection, electroporation, injection etc. Cells lacking anintact sak gene may then be identified, for example by Southernblotting, Northern Blotting or by assaying for expression of Sak proteinusing the methods described herein. Such cells may then be fused toembryonic stem cells to generate transgenic non-human animals deficientin Sak. Germline transmission of the mutation may be achieved, forexample, by aggregating the embryonic stem cells with early stageembryos, such as 8 cell embryos, in vitro; transferring the resultingblastocysts into recipient females and; generating germline transmissionof the resulting aggregation chimeras. Such a mutant animal may be usedto define specific cell populations, developmental patterns and in vivoprocesses, normally dependent on Sak expression.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES

The following materials and methods were utilized in the investigationsoutlined in the examples:

cDNA Cloning and Molecular Analysis

A cDNA library was prepared in pCDM8 using poly(A)+ RNA from D33W25, amurine lymphoid tumor cell line (Dennis, J. W. (1986) Cancer Res. 46,4594-4600). CHOP cells, a subline of CHO cells which expresses polyomalarge T (LT) antigen were transiently transfected with the cDNA libraryusing a modified DEAE-dextran procedure (Heffernan, M. & Dennis, J. W.(1991) Nucleic Acids Research 19, 85-92). After 72 hours, transfectedcells were placed in selection medium containing 50 μg/ml of thecytotoxic lectin wheat germ agglutinin (WGA), and 3 weeks later plasmidDNA was recovered from the WGA resistant clones by the Hirt procedure(Hirt, B. (1967) J. Mol. Biol. 26, 365-369). Radiolabelled probes wereprepared by random priming using the T7 Quick Prime kit (Pharmacia), andthe dideoxynucleotide DNA sequencing method was used according to themanufacturers instructions (United States Biochemical Corporation). cDNAsequences were compared to the GenBank and EMBL databases using theFASTA program and amino acid alignments were done using the MALIGNEDprogram, both from the University of Wisconsin Genetics Computer Group.

In situ RNA Analysis

In situ RNA hybridization was carried out on 8-10 μm cryostat sections,as described in Peter, M., Nakagawa, J., Dotes, M., Labbe, J. C. & Nigg,E. A. (1990) Cell 60, 791-801. Adjacent sections were probed withantisense sak-a and sak-b. Control probes including a sak-a sense probe,the neuron-specific marker SCG10, as well as c-kit and Steel; the latterthree probes gave specific non-overlapping patterns (data not shown).The 1.5 kb 17S fragment, unique to the sak-a transcript, was subclonedinto pBSK (Stratagene) and antisense probes were generated by T7 RNApolymerase (probe 1, FIG. 1). To generate a sak-b specific probe the3'end EcoRI/XhoI fragment (1.5 kb) was labelled with T3 RNA polymerase(probe 3, FIG. 1). Post-hybridization washings included treatment with50 μg/ml RNase A at 37° C. or 42° C. for 30 minutes, and 2 stringentwashes of 20 minutes each at 60° C. in 0.1X SSC. The slides were dippedin Kodak NTB-2 emulsion, exposed for 4-6 days, developed and stainedwith toluidine blue.

Sense and Anti-Sense sak Expression Vectors

The hygromycin resistance gene driven by the β-actin promoter wasexcised from the pSP-β-hygroA vector (gift from Cecilia Moens and Dr. J.Rossant, Mount Sinai Hospital) by an EcoRV/PvuII digest and cloned intothe ScaI site of pCDM8 vectors containing either no insert (control) orsense and antisense sak cDNA fragments under the control of the CMVpromoter. For the colony formation assay, 30 μg of each plasmid waslinearized by ClaI, HindIII or SacI and electroporated into 5×10⁶ CHOcells. Colonies were selected in α-MEM with 10% FCS and 400 μg/mlhygromycin B for 10 days. Colonies were stained in 0.06% methylene blueand 1.25% glutaraldehyde in PBS for 1 hour and then counted.

Example 1 Isolation of Murine sak cDNAs

In an attempt to clone genes regulating sialylation, a murine lymphoidcDNA library was transfected into CHOP cells, followed three days laterby the addition of the sialic acid-binding lectin, WGA. From thisscreen, four WGA resistant CHOP clones retained episomal plasmidcarrying an identical 1.5 kb cDNA insert (17S clone, FIG. 1),corresponding to a partial fragment of the sak-a gene (see below) in anantisense orientation. However, the relationship between theundersialylated phenotype and sak expression remains to be determinedsince transient transfections of CHOP cells with an expression vectorcontaining this antisense fragment did not result in a high frequency ofWGA-resistant colonies (data not shown).

Additional cDNA clones were isolated from the lymphoid cDNA libraryusing the 17S partial cDNA fragment as a probe (probe 1, FIG. 1), andsubsequently full-length clones were obtained by screening the librarywith a more 5' fragment (probe 2, FIG. 1). Sequence analysis of theisolated clones identified two transcriptional units, designated sak-aand sak-b (FIG. 1). The sak-a and sak-b cDNA likely representalternatively spliced forms of the gene given that the sequences divergeafter an AG dinucleotide (nt 1456, 1457), a sequence frequently found 5'to splice-donor sites (Padgett, R. A., Grabowski, P. J., Konarska, M.M., Seller, S. & Sharp, P. A. (1986) Ann. Rev. Biochem. 55, 1119-1150).A 205 nt 5'untranslated region precedes the predicted start site oftranslation, with the AACATGG sequence surrounding the intitation sitematching closely to the Kozak consensus for intiator methionines (Kozak,M. (1986) Cell 44, 283-292). The sak-a transcriptional unit encodes a925 amino acid protein (M_(r) =103 kDa) and the sak-b encoded protein is464 amino acids in length (M_(r) =53 kDa), with the first 416 aminoacids of both proteins being identical but each having differentC-terminal tails of 509 (FIG. 2, SEQ ID NOS:3 and 4) and 48 amino acids(FIG. 3, SEQ ID NOS:5 and respectively. The 205 nt 5'untranslated regionof sak is 71% G+C, and computer folding of this region suggests severalenergetically favourable secondary structures are possible (ie. deltaG=-89 Kcal), which may reduce translational efficiency (14). In sak-aclones, 3'untranslated regions of 52 nt or 464 nt were identified, eachcontaining a consensus polyadenylation signal (AATAAA) 22 nt and 20 ntupstream of a poly(A) tract, respectively (FIG. 2, SEQ. ID. NO.3). Thisregion is rich and contains three ATTTA sequences, features which havebeen shown to decrease the intracellular half-lives of transcripts(Shaw, G. & Kamen, R. (1986) Cell 46, 659-667). The sequence of the 3'untranslated region of the sak-b transcriptional unit is approximately 2kb in length and lacks a poly(A) consensus signal and tail, suggestingthe corresponding cDNA clones may not represent a full-length transcript(sequence not shown).

The N-terminal region common to both sak-a and sak-b proteins hassignificant homology to the polo subfamily of serine/threonine kinases,including the Drosophila polo kinase (Llamazares, S., Moreira, A.,Tavares, A., Girdham, C., Spruce, B. A., Gonzales, C., Karess, R. E.,Glover, D. M. & Sunkel, C. E. (1991) Genes & Dev. 5, 2153-2165), themurine snk protein (Simmons, D. L., Neel, B. G., Stevens, R., Evett, G.& Erikson, R. L. (1992) Mol. and Cell. Biol. 12, 4164-4169), the CDC5kinase of S. cerevisiae (Kitada, K., Johnson, A. L., Johnston, L. H. &Sugino, A. (1993) Mol. and Cell. Biol. 13, 4445-4457) and the murine plkprotean (Clay, F. J., McEwen, S. J., Bertoncello, I., Wilks, A. F. &Dunn, A. R. (1993) Proc. Natl. Acad. Sci. USA 90, 4882-4886) (ie. 42%,41%, 39% and 37% identity, respectively). In addition, these proteinshave a common structural organization with the kinase domain located inthe N-terminus, followed by a C-terminal domain of unknown function.Given that sak is most closely related to the polo subfamily of kinases,and is the third murine member of this group, it has been named sak forsnk/plk akin kinase.

An alignment of the kinase domains of sak and the polo subfamily membersis shown in FIG. 4. The original four polo subfamily members contain thesequence GXGGFAXC within subdomain I, with both the alanine and cysteineresidues being uncommon in these positions. The sak kinase contains thealanine residue of this motif, but the cysteine is replaced with valine,a residue more frequently found at this position in otherserine/threonine kinases. The sak kinase domain also divergessignificantly from other members of the polo subfamily in subdomainVI-B. Within this subdomain, the threonine residue in the sak sequenceDLTLSN is in a position normally occupied by a lysine, not only inproteins within the polo subfamily, but in the majority ofserine/threonine kinases. The serine residue in this sequence isreplaced by a glycine in all other members of the polo subfamily. Sakalso has the sequence GTPNYISPE in subdomain VIII which matches closelywith the GTXXYXAPE consensus sequence characteristic of serine/threoninekinases (Hanks, S. K. & Quinn, A. M. (1991) Methods. Enzymol. 200,38-63).

The C-terminal domains of Sak-a and Sak-b showed no significantsimilarities to each other or to any sequences found in the databank. Inaddition, a 30 amino acid homology domain identified in the C-terminusof the polo, CDC5, plk and snk kinases is absent in both of thepredicted sak proteins (Clay, F. J., McEwen, S. J., Bertoncello, I.,Wilks, A. F. & Dunn, A. R. (1993) Proc. Natl. Acad. Sci. USA 90,4882-4886). Three PEST regions, each containing 2 sequences in tandem inSak-a and one in Sak-b, were identified in the C-terminal region of theproteins (In FIG. 2, the PEST amino acid sequences are boxed with themiddle basic residue breaking the sequence in two PEST regions). PESTsequences are rich in proline, serine, threonine, aspartate andglutamate residues, and flanked on either side by a basic amino acid.They are found in a number of proteins with short intracellularhalf-lives and appear to contribute directly to message instability(Rogers, S., Wells, R. & Rechsteiner, M. (1986) Science 234, 364-368).

Example 2 Tissue Specific Expression of sak

Total RNA (10 ug) from mouse tissues was separated by agarose gelelectrophoresis, transferred to a nylon membrane and probed with eithera sak-a (17S probe, FIG. 1) or a β-actin probe. The right-most two lanesof testis RNA from 8 day and adult mice more clearly demonstrates thechange in sak-a transcript size (4.5 kb to 4.0 kb) that occurs followingpubertal maturation. Northern analysis of adult tissues showed sak-aexpression to be most abundant in the testis compared to all othertissues examined (FIG. 5). Transcript levels in the testis werecomparatively low at day 8 (is. pre-meiosis) and increased with age in amanner that reflects the increase in meiotic activity occurring at thesetime points. Interestingly, a 4.5 kb transcript was detected at day 8,while a 4 kb transcript was predominant in three week old and adulttestis (FIG. 5). 4.5 and 4.0 kb transcripts were also detected at lowerlevels in the spleen, thymus, ovary and 12.5 dpc (days postconception)embryonic RNA and not in the heart, liver, kidney or brain. Northernblots probed with sequences unique to the 3' untranslated region ofsak-b (probe 3, FIG. 1) identified transcripts of 3.5 kb and 4 kb thatwere expressed at several fold lower levels than sak-a but with the sametissue specific patterns, including the stage-specific change inexpression level and transcript size seen in the testis upon maturation(data not shown).

Example 3 Spatial Localization of sak Transcripts by in situHybridization

To analyse sak expression during embryonic development, ³⁵ S-labelledantisense probes specific to the sak-a (probe 1, FIG. 1) or sak-b (probe3, FIG. 1) transcripts were hybridized in situ to sections of embryos atdifferent gestational stages.

FIG. 6 shows the RNA in situ localization of sak-a expression duringmurine embryonic development. (A) Bright and (B) dark fieldphotomicrographs of sagittal sections through 7.5 dpc embryo embedded inthe uterus hybridized with a sak-a probe. (C) Bright and (D) dark fieldphotomicrographs of sagittal sections through a 11.5 dpc embryohybridized with a sak-a probe. (E,K,M) Bright and (F,L,N) dark fieldphotomicrographs of sagittal sections through a 17.5dpc embryohybridized with a sak-a probe. Arrowheads in (K) mark the border betweenthe olfactory and respiratory epithelia. (G) Bright fieldphotomicrograph of a coronal section through a 13.5 dpc embryo withadjacent sections probed with (H) sak-a (I) SCG10 and (J) sense sak-acontrol. 4v, fourth ventricle; ac, amniotic cavity; aq, aqueduct; bl,basal layer; de, decidua; ec, ectoplacental cone; es, esophagus; ex,exocoelom; gu, gut; he, heart; Jo, Jacobsons organ; ki, kidney; la,larynx; li, liver; lu, lung; lv, lateral ventricle; mp, maxillarypalate; nc, nasal cavity; np, nasopharynx; ns, nasal septum; ob,olfactory bulb; oe, olfactory epithelium; ol, olfactory pit; op,oropharynx; re, respiratory epithelium; sc, spinal cord; st, stomach;tb, turbinate bone; th, thymus; to, tongue; tr, trachea; ur, urogenitalridge.

FIG. 7 shows RNA in situ localization of sak expression in the adult gutand gonads. All sections were hybridized with a sak-a specific probe asdescribed herein. (A). Bright and (B) dark field photomicrographs ofsections through the ovary of a superovulated female induced by pregnantmare serum gonadotropins. gr, granulosa cell; oo, oocyte. (C) Bright and(D) dark field photomicrographs of sections through an adult testis. Le,Leydig cell; sg, spermatogonia; sp, spermatocyte; st. spermatids. (E)Bright and (F) dark field photomicrographs of sections through the adultgut. cr, crypt cells; il, intestinal lumen; sm, smooth muscle layer; vi,villi.

The expression patterns of sak-a and sak-b were similar but the relativeamount of transcripts was much higher for sak-a. At the 7.5 dpcprimitive streak stage, sak transcripts were evenly distributed in bothembryonic and extraembryonic tissues and much lower levels were detectedwithin the maternal decidua (FIG. 6B). With the onset of organogenesis,sak expression became more restricted in a manner that reflected theregionalization of proliferating zones. For example, sak expression inthe central nervous system (CNS) was confined to the ventricular zonesof the brain and spinal cord at all stages of development of thesestructures [eg. 11.5 dpc (FIG. 6D), 17.5 dpc (FIG. 6F)]. Duringembryogenesis, expression of sak was detected within many organs duringtheir proliferative stages, including the skin, liver, thymus, smallintestine, and the cortical layer of the kidney (FIGS. 6D,6F). In theadult small intestine, sak expression was restricted to the region atthe base of the crypts where cell division is occurring and could not bedetected in the epithelium lining the villi (FIG. 6F).

Interestingly, sak transcripts were particularly prominent duringembryogenesis in the nasal cavity within both the olfactory and nasalmucosa (FIGS. 6D,6F). To examine expression in the olfactory epitheliummore closely, consecutive sections cut through the head of a 13.5 dpcembryo were hybridized with sak (FIG. 6H) and SCG10 (FIG. 6I). Intransverse sections through the head of 13.5 dpc embryos, sak hybridizedto a region basal to the olfactory neuron layer identified by the neuronspecific SCG10 probe (Stein, R., Mori, N., Matthews, K., Lo, L. -C. &Anderson, D. J. (1988) Neuron 1, 463-476), suggesting that sak isexpressed in the basal layer of the olfactory epithelium and not in theepithelial or neuronal layers. In these sections, sak expression wasalso detected in the vomeronasal organ, which contains an accessoryolfactory epithelium resembling the olfactory mucosa. Control sectionshybridized with a sak sense probe did not show expression in theseregions, confirming the specificity of this expression pattern (FIG.6J). Expression in the basal cell layer of the olfactory epitheliumpersisted until at least day 17.5 pc (FIGS. 6F, 6L). In addition, at day17.5 pc high levels of sak transcripts were detected in the epitheliumlining the nonneuronal portion of the nasal cavity (FIGS. 6F, 6L) anexpression pattern which continued into the similarly structuredepithelial layer of the upper respiratory tract, bronchi and largebronchioles (FIGS. 6F, 6N).

In sections through the testis the highest levels of expression were inthe meiotically dividing spermatocytes and much lower levels were seenin the spermatogonia, the immediate post-meiotic round spermatids andmature spermatozoa (FIG. 7D). No expression was detected in theepithelial Sertoli cells or stromal Leydig cells. Examination ofgonadotropin superovulated ovaries at different stages of the cyclerevealed high levels of sak expression in oocytes during all phases ofgrowth. Lower levels of sak transcripts were also detected within thegranulosa cells after the initiation of follicular growth by pregnantmare serum gonadotropins, with expression levels correlating with theproliferative state.

Example 4 Expression of a sak-a Antisense Fragment Suppressed CellProliferation

To determine whether sak expression is required for cell growth, anantisense approach was used to examine the effect of decreasingexpression of this gone on the ability of CHO cells to form colonies.Colony formation has been used to assess the ability of cyclin D1(Quelle, D. E., Ashmun, R. A., Shurtleff, S. A., Karo, J-y., Bar-Sagi,D., Roussel, M. P. & Sherr, C. J. (1993) Genes & Dev. 7, 1559-1571) aswell as the p53 (Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J.K. V. & Vogelstein, B. (1993) Science 249, 912-915) and retinoblastoma(Qin, x-q., Chittendon, T., Livingston, D. M. & Kaelin, W. G. Jr. (1992)Genes & Dev. 6, 953-964) tumor suppressor genes to suppress cell growth.Growth suppression was measured by transfecting CHO cells with a vectorexpressing both the antisense sak-a (17S) fragment and the hygromycin Bresistance gone. In particular, CHO cells were transfected with pCDM8vectors containing cDNA inserts under the control of the CMV promoterand the hygromycin resistance cassette. The ClaI digests linearized thevector, and the HindIII cut the vector between the CMV promoter and theantisense sak cDNA to prevent expression of this fragment. Transfectedcells were selected in hygromycin B for 10 days and then enumerated. Theresults are expressed as a percentage of colony formation in culturestransfected with construct #1, which produced 318, 239, 159 colonies per5×10⁵ transfected cells in 3 independent experiments. Antisense sak-aexpression decreased the efficiency of colony formation in the presenceof hygromycin B by twenty-fold compared to cells transfected with vectorcontaining only the hygromycin resistance gone, whereas expression ofsense sak-a or sak-b had little or no effect on colony formation (Table1). In addition, digesting the antisense construct between the CMVpromoter and the 17S insert with HindIII (Table 1) or SacI (data notshown) restored colony formation to normal levels, demonstrating thatantisense expression is responsible for the inhibition of cell growth.

Having illustrated and described the principles of the invention in apreferred embodiment, it should be appreciated to those skilled in theart that the invention can be modified in arrangement and detail withoutdeparture from such principles. We claim all modifications coming withinthe scope of the following claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

The following sequence listings form part of the application.

                  TABLE 1                                                         ______________________________________                                        Suppression of Colony Formation by Antisense sak-a                                                   % hygromycin B-                                        Construct   Digestion  resistant colonies                                                                        n                                          ______________________________________                                        No Sak insert                                                                             Cla I      100         3                                          Sak-a antisense                                                                           Cla 1      4.2 +/- 1.6 3                                          Sak-a antisense                                                                           Hind III   87          1                                          Sak-a sense Cla I      85          1                                          Sak-b sense Cla I      82          1                                          ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 14                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1453 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (D) DEVELOPMENTAL STAGE: Lymphoid cDNA Library                                (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Murine Lymphoid                                                  (B) CLONE: WGA-Resistant Chop Clones                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 206..1453                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..205                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCGGGAATTTTTCAAAATGGGAGCTCCGGGGCGCCGCCCAGGCCTCGGGAGGTACCGGGG60                GTACCTTTCGGTGGCGTTGGCGGCGTCGCCAGCGGCGGCGTAGAGAAGGCGTCCTGATGG120               GCGCCAAGACCTGCTGGCTTCTCGGAGCGCTGCCTCGGAGGGGACTGCGAGAAGGCCGAG180               CCCCGGGCGCCGGCGGCTCGGGAACATGGCGGCGTGCATCGGGGAGAGGATC232                       MetAlaAlaCysIleGlyGluArgIle                                                   15                                                                            GAGGACTTTAAGGTTGGAAATCTACTCGGTAAAGGATCATTTGCTGGT280                           GluAspPheLysValGlyAsnLeuLeuGlyLysGlySerPheAlaGly                              10152025                                                                      GTCTACAGAGCTGAGTCCATACACACTGGTTTGGAAGTTGCAATCAAA328                           ValTyrArgAlaGluSerIleHisThrGlyLeuGluValAlaIleLys                              303540                                                                        ATGATAGATAAGAAAGCCATGTACAAAGCTGGAATGGTACAGAGAGTC376                           MetIleAspLysLysAlaMetTyrLysAlaGlyMetValGlnArgVal                              455055                                                                        CAAAATGAGGTGAAAATACATTGCCAGTTGAAACACCCCTCTGTCTTG424                           GlnAsnGluValLysIleHisCysGlnLeuLysHisProSerValLeu                              606570                                                                        GAGCTCTATAATTACTTTGAAGATAACAATTATGTCTACCTGGTATTG472                           GluLeuTyrAsnTyrPheGluAspAsnAsnTyrValTyrLeuValLeu                              758085                                                                        GAAATGTGCCACAATGGAGAAATGAACAGATATCTGAAGAACAGAATG520                           GluMetCysHisAsnGlyGluMetAsnArgTyrLeuLysAsnArgMet                              9095100105                                                                    AAGCCTTTCTCAGAAAGGGAAGCTAGGCACTTCATGCACCAGATTATC568                           LysProPheSerGluArgGluAlaArgHisPheMetHisGlnIleIle                              110115120                                                                     ACAGGAATGTTATATCTTCATTCTCATGGCATATTGCACCGGGACCTC616                           ThrGlyMetLeuTyrLeuHisSerHisGlyIleLeuHisArgAspLeu                              125130135                                                                     ACACTCTCTAACATCTTACTTACGCGGAATATGAACATAAAAATTGCT664                           ThrLeuSerAsnIleLeuLeuThrArgAsnMetAsnIleLysIleAla                              140145150                                                                     GACTTTGGACTAGCAACGCAGTTGAATATGCCACATGAAAAGCACTAT712                           AspPheGlyLeuAlaThrGlnLeuAsnMetProHisGluLysHisTyr                              155160165                                                                     ACACTCTGTGGGACTCCTAATTATATTTCACCAGAAATTGCAACTCGA760                           ThrLeuCysGlyThrProAsnTyrIleSerProGluIleAlaThrArg                              170175180185                                                                  AGTGCACATGGACTTGAATCTGATATTTGGTCATTGGGCTGTATGTCT808                           SerAlaHisGlyLeuGluSerAspIleTrpSerLeuGlyCysMetSer                              190195200                                                                     TATACGTTACTTATTGGAAGACCACCTTTTGACACTGACACAGTCAAG856                           TyrThrLeuLeuIleGlyArgProProPheAspThrAspThrValLys                              205210215                                                                     AACACATTGAACAAAGTAGTCCTGGCAGATTATGAAATGCCAGCCTTT904                           AsnThrLeuAsnLysValValLeuAlaAspTyrGluMetProAlaPhe                              220225230                                                                     TTGTCACGAGAGGCCCAGGACCTTATCCACCAGTTACTTCGTAGAAAC952                           LeuSerArgGluAlaGlnAspLeuIleHisGlnLeuLeuArgArgAsn                              235240245                                                                     CCTGCAGATCGGTTAAGTCTGTCTTCTGTGTTGGACCATCCTTTCATG1000                          ProAlaAspArgLeuSerLeuSerSerValLeuAspHisProPheMet                              250255260265                                                                  TCACGAAATCCTTCACCAAAGAGTAAAGACGTAGGGACTGTAGAGGAC1048                          SerArgAsnProSerProLysSerLysAspValGlyThrValGluAsp                              270275280                                                                     TCAATGGATAGTGGGCATGCTACACTTTCCACAACAATTACAGCCTCT1096                          SerMetAspSerGlyHisAlaThrLeuSerThrThrIleThrAlaSer                              285290295                                                                     TCTGGTACCAGTTTGAGTGGCAGCCTACTTGACAGAAGACTTTTGGTT1144                          SerGlyThrSerLeuSerGlySerLeuLeuAspArgArgLeuLeuVal                              300305310                                                                     GGTCAACCACTTCCAAATAAAATTACTGTATTTCAAAAAAATAAAAAT1192                          GlyGlnProLeuProAsnLysIleThrValPheGlnLysAsnLysAsn                              315320325                                                                     TCAAGTGACTTTTCTTCAGGAGATGGAAGTAATTTTTGTACTCAATGG1240                          SerSerAspPheSerSerGlyAspGlySerAsnPheCysThrGlnTrp                              330335340345                                                                  GGAAATCCAGAACAAGAAGCTAATAGTAGGGGACGGGGGAGAGTGATT1288                          GlyAsnProGluGlnGluAlaAsnSerArgGlyArgGlyArgValIle                              350355360                                                                     GAAGATGCAGAAGAGAGGCCGCATTCTCGATACCTGCGCAGAGCTCAT1336                          GluAspAlaGluGluArgProHisSerArgTyrLeuArgArgAlaHis                              365370375                                                                     TCCTCTGATAGAGCCAGCCCCTCTAATCAGTCTCGAGCAAAAACATAC1384                          SerSerAspArgAlaSerProSerAsnGlnSerArgAlaLysThrTyr                              380385390                                                                     TCAGTAGAACGTTGTCACTCAGTAGAAATGCTTTCAAAGCCTAGAAGA1432                          SerValGluArgCysHisSerValGluMetLeuSerLysProArgArg                              395400405                                                                     TCACTGGATGAAAATCAACAC1453                                                     SerLeuAspGluAsnGlnHis                                                         410415                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 416 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaAlaCysIleGlyGluArgIleGluAspPheLysValGlyAsn                              151015                                                                        LeuLeuGlyLysGlySerPheAlaGlyValTyrArgAlaGluSerIle                              202530                                                                        HisThrGlyLeuGluValAlaIleLysMetIleAspLysLysAlaMet                              354045                                                                        TyrLysAlaGlyMetValGlnArgValGlnAsnGluValLysIleHis                              505560                                                                        CysGlnLeuLysHisProSerValLeuGluLeuTyrAsnTyrPheGlu                              65707580                                                                      AspAsnAsnTyrValTyrLeuValLeuGluMetCysHisAsnGlyGlu                              859095                                                                        MetAsnArgTyrLeuLysAsnArgMetLysProPheSerGluArgGlu                              100105110                                                                     AlaArgHisPheMetHisGlnIleIleThrGlyMetLeuTyrLeuHis                              115120125                                                                     SerHisGlyIleLeuHisArgAspLeuThrLeuSerAsnIleLeuLeu                              130135140                                                                     ThrArgAsnMetAsnIleLysIleAlaAspPheGlyLeuAlaThrGln                              145150155160                                                                  LeuAsnMetProHisGluLysHisTyrThrLeuCysGlyThrProAsn                              165170175                                                                     TyrIleSerProGluIleAlaThrArgSerAlaHisGlyLeuGluSer                              180185190                                                                     AspIleTrpSerLeuGlyCysMetSerTyrThrLeuLeuIleGlyArg                              195200205                                                                     ProProPheAspThrAspThrValLysAsnThrLeuAsnLysValVal                              210215220                                                                     LeuAlaAspTyrGluMetProAlaPheLeuSerArgGluAlaGlnAsp                              225230235240                                                                  LeuIleHisGlnLeuLeuArgArgAsnProAlaAspArgLeuSerLeu                              245250255                                                                     SerSerValLeuAspHisProPheMetSerArgAsnProSerProLys                              260265270                                                                     SerLysAspValGlyThrValGluAspSerMetAspSerGlyHisAla                              275280285                                                                     ThrLeuSerThrThrIleThrAlaSerSerGlyThrSerLeuSerGly                              290295300                                                                     SerLeuLeuAspArgArgLeuLeuValGlyGlnProLeuProAsnLys                              305310315320                                                                  IleThrValPheGlnLysAsnLysAsnSerSerAspPheSerSerGly                              325330335                                                                     AspGlySerAsnPheCysThrGlnTrpGlyAsnProGluGlnGluAla                              340345350                                                                     AsnSerArgGlyArgGlyArgValIleGluAspAlaGluGluArgPro                              355360365                                                                     HisSerArgTyrLeuArgArgAlaHisSerSerAspArgAlaSerPro                              370375380                                                                     SerAsnGlnSerArgAlaLysThrTyrSerValGluArgCysHisSer                              385390395400                                                                  ValGluMetLeuSerLysProArgArgSerLeuAspGluAsnGlnHis                              405410415                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3447 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (D) DEVELOPMENTAL STAGE: Lymphoid cDNA Library                                (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Murine Lymphoid                                                  (B) CLONE: WGA-resistant chop clones                                          (ix) FEATURE:                                                                 (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..205                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 206..2980                                                       (ix) FEATURE:                                                                 (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 2981..3447                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCGGGAATTTTTCAAAATGGGAGCTCCGGGGCGCCGCCCAGGCCTCGGGAGGTACCGGGG60                GTACCTTTCGGTGGCGTTGGCGGCGTCGCCAGCGGCGGCGTAGAGAAGGCGTCCTGATGG120               GCGCCAAGACCTGCTGGCTTCTCGGAGCGCTGCCTCGGAGGGGACTGCGAGAAGGCCGAG180               CCCCGGGCGCCGGCGGCTCGGGAACATGGCGGCGTGCATCGGGGAGAGGATC232                       MetAlaAlaCysIleGlyGluArgIle                                                   15                                                                            GAGGACTTTAAGGTTGGAAATCTACTCGGTAAAGGATCATTTGCTGGT280                           GluAspPheLysValGlyAsnLeuLeuGlyLysGlySerPheAlaGly                              10152025                                                                      GTCTACAGAGCTGAGTCCATACACACTGGTTTGGAAGTTGCAATCAAA328                           ValTyrArgAlaGluSerIleHisThrGlyLeuGluValAlaIleLys                              303540                                                                        ATGATAGATAAGAAAGCCATGTACAAAGCTGGAATGGTACAGAGAGTC376                           MetIleAspLysLysAlaMetTyrLysAlaGlyMetValGlnArgVal                              455055                                                                        CAAAATGAGGTGAAAATACATTGCCAGTTGAAACACCCCTCTGTCTTG424                           GlnAsnGluValLysIleHisCysGlnLeuLysHisProSerValLeu                              606570                                                                        GAGCTCTATAATTACTTTGAAGATAACAATTATGTCTACCTGGTATTG472                           GluLeuTyrAsnTyrPheGluAspAsnAsnTyrValTyrLeuValLeu                              758085                                                                        GAAATGTGCCACAATGGAGAAATGAACAGATATCTGAAGAACAGAATG520                           GluMetCysHisAsnGlyGluMetAsnArgTyrLeuLysAsnArgMet                              9095100105                                                                    AAGCCTTTCTCAGAAAGGGAAGCTAGGCACTTCATGCACCAGATTATC568                           LysProPheSerGluArgGluAlaArgHisPheMetHisGlnIleIle                              110115120                                                                     ACAGGAATGTTATATCTTCATTCTCATGGCATATTGCACCGGGACCTC616                           ThrGlyMetLeuTyrLeuHisSerHisGlyIleLeuHisArgAspLeu                              125130135                                                                     ACACTCTCTAACATCTTACTTACGCGGAATATGAACATAAAAATTGCT664                           ThrLeuSerAsnIleLeuLeuThrArgAsnMetAsnIleLysIleAla                              140145150                                                                     GACTTTGGACTAGCAACGCAGTTGAATATGCCACATGAAAAGCACTAT712                           AspPheGlyLeuAlaThrGlnLeuAsnMetProHisGluLysHisTyr                              155160165                                                                     ACACTCTGTGGGACTCCTAATTATATTTCACCAGAAATTGCAACTCGA760                           ThrLeuCysGlyThrProAsnTyrIleSerProGluIleAlaThrArg                              170175180185                                                                  AGTGCACATGGACTTGAATCTGATATTTGGTCATTGGGCTGTATGTCT808                           SerAlaHisGlyLeuGluSerAspIleTrpSerLeuGlyCysMetSer                              190195200                                                                     TATACGTTACTTATTGGAAGACCACCTTTTGACACTGACACAGTCAAG856                           TyrThrLeuLeuIleGlyArgProProPheAspThrAspThrValLys                              205210215                                                                     AACACATTGAACAAAGTAGTCCTGGCAGATTATGAAATGCCAGCCTTT904                           AsnThrLeuAsnLysValValLeuAlaAspTyrGluMetProAlaPhe                              220225230                                                                     TTGTCACGAGAGGCCCAGGACCTTATCCACCAGTTACTTCGTAGAAAC952                           LeuSerArgGluAlaGlnAspLeuIleHisGlnLeuLeuArgArgAsn                              235240245                                                                     CCTGCAGATCGGTTAAGTCTGTCTTCTGTGTTGGACCATCCTTTCATG1000                          ProAlaAspArgLeuSerLeuSerSerValLeuAspHisProPheMet                              250255260265                                                                  TCACGAAATCCTTCACCAAAGAGTAAAGACGTAGGGACTGTAGAGGAC1048                          SerArgAsnProSerProLysSerLysAspValGlyThrValGluAsp                              270275280                                                                     TCAATGGATAGTGGGCATGCTACACTTTCCACAACAATTACAGCCTCT1096                          SerMetAspSerGlyHisAlaThrLeuSerThrThrIleThrAlaSer                              285290295                                                                     TCTGGTACCAGTTTGAGTGGCAGCCTACTTGACAGAAGACTTTTGGTT1144                          SerGlyThrSerLeuSerGlySerLeuLeuAspArgArgLeuLeuVal                              300305310                                                                     GGTCAACCACTTCCAAATAAAATTACTGTATTTCAAAAAAATAAAAAT1192                          GlyGlnProLeuProAsnLysIleThrValPheGlnLysAsnLysAsn                              315320325                                                                     TCAAGTGACTTTTCTTCAGGAGATGGAAGTAATTTTTGTACTCAATGG1240                          SerSerAspPheSerSerGlyAspGlySerAsnPheCysThrGlnTrp                              330335340345                                                                  GGAAATCCAGAACAAGAAGCTAATAGTAGGGGACGGGGGAGAGTGATT1288                          GlyAsnProGluGlnGluAlaAsnSerArgGlyArgGlyArgValIle                              350355360                                                                     GAAGATGCAGAAGAGAGGCCGCATTCTCGATACCTGCGCAGAGCTCAT1336                          GluAspAlaGluGluArgProHisSerArgTyrLeuArgArgAlaHis                              365370375                                                                     TCCTCTGATAGAGCCAGCCCCTCTAATCAGTCTCGAGCAAAAACATAC1384                          SerSerAspArgAlaSerProSerAsnGlnSerArgAlaLysThrTyr                              380385390                                                                     TCAGTAGAACGTTGTCACTCAGTAGAAATGCTTTCAAAGCCTAGAAGA1432                          SerValGluArgCysHisSerValGluMetLeuSerLysProArgArg                              395400405                                                                     TCACTGGATGAAAATCAACACAGTTCCAATCATCATTGTCTAGGAAAA1480                          SerLeuAspGluAsnGlnHisSerSerAsnHisHisCysLeuGlyLys                              410415420425                                                                  ACTCCTTTTCCATTTGCAGACCAGACACCTCAGATGGAAATGGTACAG1528                          ThrProPheProPheAlaAspGlnThrProGlnMetGluMetValGln                              430435440                                                                     CAGTGGTTTGGGAATCTGCAAATGAATGCTCATTTAGGAGAAACTAAT1576                          GlnTrpPheGlyAsnLeuGlnMetAsnAlaHisLeuGlyGluThrAsn                              445450455                                                                     GAGCACCACACCGTTAGCCCAAACAGAGATTTCCAGGACTATCCAGAT1624                          GluHisHisThrValSerProAsnArgAspPheGlnAspTyrProAsp                              460465470                                                                     TTGCAGGACACGTTACGAAACGCTTGGACTGACACGAGAGCCAGCAAG1672                          LeuGlnAspThrLeuArgAsnAlaTrpThrAspThrArgAlaSerLys                              475480485                                                                     AATGCTGATACTTCTGCCAATGTTCATGCTGTAAAGCAGCTGAGTGCC1720                          AsnAlaAspThrSerAlaAsnValHisAlaValLysGlnLeuSerAla                              490495500505                                                                  ATGAAATACATGAGTGCACATCACCATAAGCCTGAGGTCATGCCACAG1768                          MetLysTyrMetSerAlaHisHisHisLysProGluValMetProGln                              510515520                                                                     GAGCCGGGCCTACATCCTCATTCTGAACAAAGCAAGAATAGAAGTATG1816                          GluProGlyLeuHisProHisSerGluGlnSerLysAsnArgSerMet                              525530535                                                                     GAGTCGACACTGGGTTACCAGAAACCTACCTTAAGAAGTATTACATCT1864                          GluSerThrLeuGlyTyrGlnLysProThrLeuArgSerIleThrSer                              540545550                                                                     CCTCTGATTGCTCACAGATTAAAGCCAATCAGACAGAAAACCAAAAAG1912                          ProLeuIleAlaHisArgLeuLysProIleArgGlnLysThrLysLys                              555560565                                                                     GCTGTGGTGAGCATCCTTGATTCAGAGGAGGTGTGTGTGGAGCTTCTG1960                          AlaValValSerIleLeuAspSerGluGluValCysValGluLeuLeu                              570575580585                                                                  AGAGAGTGTGCGTCTGAAGGATATGTGAAAGAAGTGCTTCAGATATCG2008                          ArgGluCysAlaSerGluGlyTyrValLysGluValLeuGlnIleSer                              590595600                                                                     AGTGATGGGACTATGATCACTGTTTATTACCCGAACGATGGAAGAGGC2056                          SerAspGlyThrMetIleThrValTyrTyrProAsnAspGlyArgGly                              605610615                                                                     TTTCCTCTTGCTGACAGACCTCCCTTGCCTACTGACAACATCAGTAGG2104                          PheProLeuAlaAspArgProProLeuProThrAspAsnIleSerArg                              620625630                                                                     TACAGCTTTGACAATCTACCAGAAAAATACTGGCGGAAATATCAGTAT2152                          TyrSerPheAspAsnLeuProGluLysTyrTrpArgLysTyrGlnTyr                              635640645                                                                     GCTTCCAGATTCATTCAGCTAGTAAGATCTAAAACTCCCAAAATCACT2200                          AlaSerArgPheIleGlnLeuValArgSerLysThrProLysIleThr                              650655660665                                                                  TATTTTACAAGATATGCTAAATGTATTTTGATGGAAAATTCTCCTGGT2248                          TyrPheThrArgTyrAlaLysCysIleLeuMetGluAsnSerProGly                              670675680                                                                     GCTGATTTCGAAGTTTGGTTTTATGATGGAGCCAAAATACATAAAACT2296                          AlaAspPheGluValTrpPheTyrAspGlyAlaLysIleHisLysThr                              685690695                                                                     GAAAATTTAATTCACATAATTGAGAAAACAGGGATATCTTATAATTTA2344                          GluAsnLeuIleHisIleIleGluLysThrGlyIleSerTyrAsnLeu                              700705710                                                                     AAAAATGAAAATGAAGTTACCAGCCTGAAAGAGGAAGTAAAAGTATAT2392                          LysAsnGluAsnGluValThrSerLeuLysGluGluValLysValTyr                              715720725                                                                     ATGGACCATGCTAATGAGGGTCACCGTATTTGCTTGTCACTGGAATCT2440                          MetAspHisAlaAsnGluGlyHisArgIleCysLeuSerLeuGluSer                              730735740745                                                                  GTAATCTCTGAGGAGGAAAAGAGAAGCAGGGGTTCTTCATTCTTCCCT2488                          ValIleSerGluGluGluLysArgSerArgGlySerSerPhePhePro                              750755760                                                                     ATAATCGTAGGAAGAAAACCTGGTAATACTAGTTCACCTAAAGCCTTA2536                          IleIleValGlyArgLysProGlyAsnThrSerSerProLysAlaLeu                              765770775                                                                     TCAGCTCCTCCTGTGGACCCAAGCTGCTGTAAGGGAGAGCAGGCGTCA2584                          SerAlaProProValAspProSerCysCysLysGlyGluGlnAlaSer                              780785790                                                                     GCAAGCAGACTGAGCGTGAATAGTGCCGCTTTCCCCACACAGTCCCCA2632                          AlaSerArgLeuSerValAsnSerAlaAlaPheProThrGlnSerPro                              795800805                                                                     GGACTCAGTCCTTCCACTGTGACAGTTGAAGGACTTGGCCACACAGCG2680                          GlyLeuSerProSerThrValThrValGluGlyLeuGlyHisThrAla                              810815820825                                                                  ACTGCCACAGGAACAGGCGTCTCTTCAAGTCTTCCTAAATCTGCACAG2728                          ThrAlaThrGlyThrGlyValSerSerSerLeuProLysSerAlaGln                              830835840                                                                     CTTTTGAAATCTGTTTTTGTGAAAAATGTTGGTTGGGCTACACAGCTA2776                          LeuLeuLysSerValPheValLysAsnValGlyTrpAlaThrGlnLeu                              845850855                                                                     ACTAGCGGAGCTGTGTGGGTTCAGTTTAATGATGGGTCACAGTTGGTT2824                          ThrSerGlyAlaValTrpValGlnPheAsnAspGlySerGlnLeuVal                              860865870                                                                     GTCCAGGCAGGAGTATCTTCCATCAGTTACACATCACCAGATGGTCAG2872                          ValGlnAlaGlyValSerSerIleSerTyrThrSerProAspGlyGln                              875880885                                                                     ACAACTAGGTATGGAGAAAATGAAAAATTACCTGAATACATCAAACAG2920                          ThrThrArgTyrGlyGluAsnGluLysLeuProGluTyrIleLysGln                              890895900905                                                                  AAATTACAGTGTCTTTCTTCCATCCTTCTGATGTTTTCTAATCCAACT2968                          LysLeuGlnCysLeuSerSerIleLeuLeuMetPheSerAsnProThr                              910915920                                                                     CCTAATTTTCAGTAATTTAAGTCTCAGAAGTCTATATTTAATAAATGACTTT3020                      ProAsnPheGln                                                                  925                                                                           TTGGCTGGCTTTCAAGTAAGTGATTTTTTAAATTTACTTTAACTTCAGAAAGCCTTTCTA3080              TTAAACAGAATTTTAATATACACAATAAAAATATAATAAGAAAACAATAAAATTTCAGTT3140              ACCTAATATAGTGGTCATAAGGCTAGGACATCTAATTTTGCTCCAAGCATGTAATCCTTC3200              AAAGTTTGTGCTCCTATGTTTGTATTGAACTAAGTTGTGTATGGCTTGTTTGTTTTTGTT3260              ATTTTCTTTACTAATAAGACATTGAGAATCACGGACAAAACATAGTTTTCAATTTTTGAA3320              TGTGTAAATAATGTATTATAAGCAATATGTAAATGTGTATATTTTATATTTATTTTTATA3380              GCACTTGTGTCTGATAAGATTTCTGCAAATACATTTTATAAAATAAACACAGTGGTAAGT3440              TTTCCTT3447                                                                   (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 925 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetAlaAlaCysIleGlyGluArgIleGluAspPheLysValGlyAsn                              151015                                                                        LeuLeuGlyLysGlySerPheAlaGlyValTyrArgAlaGluSerIle                              202530                                                                        HisThrGlyLeuGluValAlaIleLysMetIleAspLysLysAlaMet                              354045                                                                        TyrLysAlaGlyMetValGlnArgValGlnAsnGluValLysIleHis                              505560                                                                        CysGlnLeuLysHisProSerValLeuGluLeuTyrAsnTyrPheGlu                              65707580                                                                      AspAsnAsnTyrValTyrLeuValLeuGluMetCysHisAsnGlyGlu                              859095                                                                        MetAsnArgTyrLeuLysAsnArgMetLysProPheSerGluArgGlu                              100105110                                                                     AlaArgHisPheMetHisGlnIleIleThrGlyMetLeuTyrLeuHis                              115120125                                                                     SerHisGlyIleLeuHisArgAspLeuThrLeuSerAsnIleLeuLeu                              130135140                                                                     ThrArgAsnMetAsnIleLysIleAlaAspPheGlyLeuAlaThrGln                              145150155160                                                                  LeuAsnMetProHisGluLysHisTyrThrLeuCysGlyThrProAsn                              165170175                                                                     TyrIleSerProGluIleAlaThrArgSerAlaHisGlyLeuGluSer                              180185190                                                                     AspIleTrpSerLeuGlyCysMetSerTyrThrLeuLeuIleGlyArg                              195200205                                                                     ProProPheAspThrAspThrValLysAsnThrLeuAsnLysValVal                              210215220                                                                     LeuAlaAspTyrGluMetProAlaPheLeuSerArgGluAlaGlnAsp                              225230235240                                                                  LeuIleHisGlnLeuLeuArgArgAsnProAlaAspArgLeuSerLeu                              245250255                                                                     SerSerValLeuAspHisProPheMetSerArgAsnProSerProLys                              260265270                                                                     SerLysAspValGlyThrValGluAspSerMetAspSerGlyHisAla                              275280285                                                                     ThrLeuSerThrThrIleThrAlaSerSerGlyThrSerLeuSerGly                              290295300                                                                     SerLeuLeuAspArgArgLeuLeuValGlyGlnProLeuProAsnLys                              305310315320                                                                  IleThrValPheGlnLysAsnLysAsnSerSerAspPheSerSerGly                              325330335                                                                     AspGlySerAsnPheCysThrGlnTrpGlyAsnProGluGlnGluAla                              340345350                                                                     AsnSerArgGlyArgGlyArgValIleGluAspAlaGluGluArgPro                              355360365                                                                     HisSerArgTyrLeuArgArgAlaHisSerSerAspArgAlaSerPro                              370375380                                                                     SerAsnGlnSerArgAlaLysThrTyrSerValGluArgCysHisSer                              385390395400                                                                  ValGluMetLeuSerLysProArgArgSerLeuAspGluAsnGlnHis                              405410415                                                                     SerSerAsnHisHisCysLeuGlyLysThrProPheProPheAlaAsp                              420425430                                                                     GlnThrProGlnMetGluMetValGlnGlnTrpPheGlyAsnLeuGln                              435440445                                                                     MetAsnAlaHisLeuGlyGluThrAsnGluHisHisThrValSerPro                              450455460                                                                     AsnArgAspPheGlnAspTyrProAspLeuGlnAspThrLeuArgAsn                              465470475480                                                                  AlaTrpThrAspThrArgAlaSerLysAsnAlaAspThrSerAlaAsn                              485490495                                                                     ValHisAlaValLysGlnLeuSerAlaMetLysTyrMetSerAlaHis                              500505510                                                                     HisHisLysProGluValMetProGlnGluProGlyLeuHisProHis                              515520525                                                                     SerGluGlnSerLysAsnArgSerMetGluSerThrLeuGlyTyrGln                              530535540                                                                     LysProThrLeuArgSerIleThrSerProLeuIleAlaHisArgLeu                              545550555560                                                                  LysProIleArgGlnLysThrLysLysAlaValValSerIleLeuAsp                              565570575                                                                     SerGluGluValCysValGluLeuLeuArgGluCysAlaSerGluGly                              580585590                                                                     TyrValLysGluValLeuGlnIleSerSerAspGlyThrMetIleThr                              595600605                                                                     ValTyrTyrProAsnAspGlyArgGlyPheProLeuAlaAspArgPro                              610615620                                                                     ProLeuProThrAspAsnIleSerArgTyrSerPheAspAsnLeuPro                              625630635640                                                                  GluLysTyrTrpArgLysTyrGlnTyrAlaSerArgPheIleGlnLeu                              645650655                                                                     ValArgSerLysThrProLysIleThrTyrPheThrArgTyrAlaLys                              660665670                                                                     CysIleLeuMetGluAsnSerProGlyAlaAspPheGluValTrpPhe                              675680685                                                                     TyrAspGlyAlaLysIleHisLysThrGluAsnLeuIleHisIleIle                              690695700                                                                     GluLysThrGlyIleSerTyrAsnLeuLysAsnGluAsnGluValThr                              705710715720                                                                  SerLeuLysGluGluValLysValTyrMetAspHisAlaAsnGluGly                              725730735                                                                     HisArgIleCysLeuSerLeuGluSerValIleSerGluGluGluLys                              740745750                                                                     ArgSerArgGlySerSerPhePheProIleIleValGlyArgLysPro                              755760765                                                                     GlyAsnThrSerSerProLysAlaLeuSerAlaProProValAspPro                              770775780                                                                     SerCysCysLysGlyGluGlnAlaSerAlaSerArgLeuSerValAsn                              785790795800                                                                  SerAlaAlaPheProThrGlnSerProGlyLeuSerProSerThrVal                              805810815                                                                     ThrValGluGlyLeuGlyHisThrAlaThrAlaThrGlyThrGlyVal                              820825830                                                                     SerSerSerLeuProLysSerAlaGlnLeuLeuLysSerValPheVal                              835840845                                                                     LysAsnValGlyTrpAlaThrGlnLeuThrSerGlyAlaValTrpVal                              850855860                                                                     GlnPheAsnAspGlySerGlnLeuValValGlnAlaGlyValSerSer                              865870875880                                                                  IleSerTyrThrSerProAspGlyGlnThrThrArgTyrGlyGluAsn                              885890895                                                                     GluLysLeuProGluTyrIleLysGlnLysLeuGlnCysLeuSerSer                              900905910                                                                     IleLeuLeuMetPheSerAsnProThrProAsnPheGln                                       915920925                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1600 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (D) DEVELOPMENTAL STAGE: Lymphoid cDNA Library                                (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Murine Lymphoid                                                  (B) CLONE: WGA-resistant chop clones                                          (ix) FEATURE:                                                                 (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..205                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 206..1597                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGGGAATTTTTCAAAATGGGAGCTCCGGGGCGCCGCCCAGGCCTCGGGAGGTACCGGGG60                GTACCTTTCGGTGGCGTTGGCGGCGTCGCCAGCGGCGGCGTAGAGAAGGCGTCCTGATGG120               GCGCCAAGACCTGCTGGCTTCTCGGAGCGCTGCCTCGGAGGGGACTGCGAGAAGGCCGAG180               CCCCGGGCGCCGGCGGCTCGGGAACATGGCGGCGTGCATCGGGGAGAGGATC232                       MetAlaAlaCysIleGlyGluArgIle                                                   15                                                                            GAGGACTTTAAGGTTGGAAATCTACTCGGTAAAGGATCATTTGCTGGT280                           GluAspPheLysValGlyAsnLeuLeuGlyLysGlySerPheAlaGly                              10152025                                                                      GTCTACAGAGCTGAGTCCATACACACTGGTTTGGAAGTTGCAATCAAA328                           ValTyrArgAlaGluSerIleHisThrGlyLeuGluValAlaIleLys                              303540                                                                        ATGATAGATAAGAAAGCCATGTACAAAGCTGGAATGGTACAGAGAGTC376                           MetIleAspLysLysAlaMetTyrLysAlaGlyMetValGlnArgVal                              455055                                                                        CAAAATGAGGTGAAAATACATTGCCAGTTGAAACACCCCTCTGTCTTG424                           GlnAsnGluValLysIleHisCysGlnLeuLysHisProSerValLeu                              606570                                                                        GAGCTCTATAATTACTTTGAAGATAACAATTATGTCTACCTGGTATTG472                           GluLeuTyrAsnTyrPheGluAspAsnAsnTyrValTyrLeuValLeu                              758085                                                                        GAAATGTGCCACAATGGAGAAATGAACAGATATCTGAAGAACAGAATG520                           GluMetCysHisAsnGlyGluMetAsnArgTyrLeuLysAsnArgMet                              9095100105                                                                    AAGCCTTTCTCAGAAAGGGAAGCTAGGCACTTCATGCACCAGATTATC568                           LysProPheSerGluArgGluAlaArgHisPheMetHisGlnIleIle                              110115120                                                                     ACAGGAATGTTATATCTTCATTCTCATGGCATATTGCACCGGGACCTC616                           ThrGlyMetLeuTyrLeuHisSerHisGlyIleLeuHisArgAspLeu                              125130135                                                                     ACACTCTCTAACATCTTACTTACGCGGAATATGAACATAAAAATTGCT664                           ThrLeuSerAsnIleLeuLeuThrArgAsnMetAsnIleLysIleAla                              140145150                                                                     GACTTTGGACTAGCAACGCAGTTGAATATGCCACATGAAAAGCACTAT712                           AspPheGlyLeuAlaThrGlnLeuAsnMetProHisGluLysHisTyr                              155160165                                                                     ACACTCTGTGGGACTCCTAATTATATTTCACCAGAAATTGCAACTCGA760                           ThrLeuCysGlyThrProAsnTyrIleSerProGluIleAlaThrArg                              170175180185                                                                  AGTGCACATGGACTTGAATCTGATATTTGGTCATTGGGCTGTATGTCT808                           SerAlaHisGlyLeuGluSerAspIleTrpSerLeuGlyCysMetSer                              190195200                                                                     TATACGTTACTTATTGGAAGACCACCTTTTGACACTGACACAGTCAAG856                           TyrThrLeuLeuIleGlyArgProProPheAspThrAspThrValLys                              205210215                                                                     AACACATTGAACAAAGTAGTCCTGGCAGATTATGAAATGCCAGCCTTT904                           AsnThrLeuAsnLysValValLeuAlaAspTyrGluMetProAlaPhe                              220225230                                                                     TTGTCACGAGAGGCCCAGGACCTTATCCACCAGTTACTTCGTAGAAAC952                           LeuSerArgGluAlaGlnAspLeuIleHisGlnLeuLeuArgArgAsn                              235240245                                                                     CCTGCAGATCGGTTAAGTCTGTCTTCTGTGTTGGACCATCCTTTCATG1000                          ProAlaAspArgLeuSerLeuSerSerValLeuAspHisProPheMet                              250255260265                                                                  TCACGAAATCCTTCACCAAAGAGTAAAGACGTAGGGACTGTAGAGGAC1048                          SerArgAsnProSerProLysSerLysAspValGlyThrValGluAsp                              270275280                                                                     TCAATGGATAGTGGGCATGCTACACTTTCCACAACAATTACAGCCTCT1096                          SerMetAspSerGlyHisAlaThrLeuSerThrThrIleThrAlaSer                              285290295                                                                     TCTGGTACCAGTTTGAGTGGCAGCCTACTTGACAGAAGACTTTTGGTT1144                          SerGlyThrSerLeuSerGlySerLeuLeuAspArgArgLeuLeuVal                              300305310                                                                     GGTCAACCACTTCCAAATAAAATTACTGTATTTCAAAAAAATAAAAAT1192                          GlyGlnProLeuProAsnLysIleThrValPheGlnLysAsnLysAsn                              315320325                                                                     TCAAGTGACTTTTCTTCAGGAGATGGAAGTAATTTTTGTACTCAATGG1240                          SerSerAspPheSerSerGlyAspGlySerAsnPheCysThrGlnTrp                              330335340345                                                                  GGAAATCCAGAACAAGAAGCTAATAGTAGGGGACGGGGGAGAGTGATT1288                          GlyAsnProGluGlnGluAlaAsnSerArgGlyArgGlyArgValIle                              350355360                                                                     GAAGATGCAGAAGAGAGGCCGCATTCTCGATACCTGCGCAGAGCTCAT1336                          GluAspAlaGluGluArgProHisSerArgTyrLeuArgArgAlaHis                              365370375                                                                     TCCTCTGATAGAGCCAGCCCCTCTAATCAGTCTCGAGCAAAAACATAC1384                          SerSerAspArgAlaSerProSerAsnGlnSerArgAlaLysThrTyr                              380385390                                                                     TCAGTAGAACGTTGTCACTCAGTAGAAATGCTTTCAAAGCCTAGAAGA1432                          SerValGluArgCysHisSerValGluMetLeuSerLysProArgArg                              395400405                                                                     TCACTGGATGAAAATCAACACAGGTATTCACCCACCAAAAGCAATGTC1480                          SerLeuAspGluAsnGlnHisArgTyrSerProThrLysSerAsnVal                              410415420425                                                                  AATGTTTTAACTTCATTAAACACCAAACAGCCAATAGTTAAGGATCTT1528                          AsnValLeuThrSerLeuAsnThrLysGlnProIleValLysAspLeu                              430435440                                                                     TTGAAAGACCGTATAATGACTGAGCAGTATAAGGATAATCTTTTAAAC1576                          LeuLysAspArgIleMetThrGluGlnTyrLysAspAsnLeuLeuAsn                              445450455                                                                     TTATTGAACAAGTTTGATCGCTAA1600                                                  LeuLeuAsnLysPheAspArg                                                         460                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 464 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAlaAlaCysIleGlyGluArgIleGluAspPheLysValGlyAsn                              151015                                                                        LeuLeuGlyLysGlySerPheAlaGlyValTyrArgAlaGluSerIle                              202530                                                                        HisThrGlyLeuGluValAlaIleLysMetIleAspLysLysAlaMet                              354045                                                                        TyrLysAlaGlyMetValGlnArgValGlnAsnGluValLysIleHis                              505560                                                                        CysGlnLeuLysHisProSerValLeuGluLeuTyrAsnTyrPheGlu                              65707580                                                                      AspAsnAsnTyrValTyrLeuValLeuGluMetCysHisAsnGlyGlu                              859095                                                                        MetAsnArgTyrLeuLysAsnArgMetLysProPheSerGluArgGlu                              100105110                                                                     AlaArgHisPheMetHisGlnIleIleThrGlyMetLeuTyrLeuHis                              115120125                                                                     SerHisGlyIleLeuHisArgAspLeuThrLeuSerAsnIleLeuLeu                              130135140                                                                     ThrArgAsnMetAsnIleLysIleAlaAspPheGlyLeuAlaThrGln                              145150155160                                                                  LeuAsnMetProHisGluLysHisTyrThrLeuCysGlyThrProAsn                              165170175                                                                     TyrIleSerProGluIleAlaThrArgSerAlaHisGlyLeuGluSer                              180185190                                                                     AspIleTrpSerLeuGlyCysMetSerTyrThrLeuLeuIleGlyArg                              195200205                                                                     ProProPheAspThrAspThrValLysAsnThrLeuAsnLysValVal                              210215220                                                                     LeuAlaAspTyrGluMetProAlaPheLeuSerArgGluAlaGlnAsp                              225230235240                                                                  LeuIleHisGlnLeuLeuArgArgAsnProAlaAspArgLeuSerLeu                              245250255                                                                     SerSerValLeuAspHisProPheMetSerArgAsnProSerProLys                              260265270                                                                     SerLysAspValGlyThrValGluAspSerMetAspSerGlyHisAla                              275280285                                                                     ThrLeuSerThrThrIleThrAlaSerSerGlyThrSerLeuSerGly                              290295300                                                                     SerLeuLeuAspArgArgLeuLeuValGlyGlnProLeuProAsnLys                              305310315320                                                                  IleThrValPheGlnLysAsnLysAsnSerSerAspPheSerSerGly                              325330335                                                                     AspGlySerAsnPheCysThrGlnTrpGlyAsnProGluGlnGluAla                              340345350                                                                     AsnSerArgGlyArgGlyArgValIleGluAspAlaGluGluArgPro                              355360365                                                                     HisSerArgTyrLeuArgArgAlaHisSerSerAspArgAlaSerPro                              370375380                                                                     SerAsnGlnSerArgAlaLysThrTyrSerValGluArgCysHisSer                              385390395400                                                                  ValGluMetLeuSerLysProArgArgSerLeuAspGluAsnGlnHis                              405410415                                                                     ArgTyrSerProThrLysSerAsnValAsnValLeuThrSerLeuAsn                              420425430                                                                     ThrLysGlnProIleValLysAspLeuLeuLysAspArgIleMetThr                              435440445                                                                     GluGlnTyrLysAspAsnLeuLeuAsnLeuLeuAsnLysPheAspArg                              450455460                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AspLeuThrLeuSerAsn                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlyThrProAsnTyrIleSerProGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyThrXaaXaaTyrXaaAlaProGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 273 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      IleGlyGluArgIleGluAspPheLysValGlyAsnLeuLeuGlyLys                              151015                                                                        GlySerPheAlaGlyValTyrArgAlaGluSerIleHisThrGlyLeu                              202530                                                                        GluValAlaIleLysMetIleAspLysLysAlaMetTyrLysAlaGly                              354045                                                                        MetValGlnArgValGlnAsnGluValLysIleHisCysGlnLeuLys                              505560                                                                        HisProSerValLeuGluLeuTyrAsnTyrPheGluAspAsnAsnTyr                              65707580                                                                      ValTyrLeuValLeuGluMetCysHisAsnGlyGluMetAsnArgTyr                              859095                                                                        LeuLysAsnArgMetLysProPheSerGluArgGluAlaArgHisPhe                              100105110                                                                     MetHisGlnIleIleThrGlyMetLeuTyrLeuHisSerHisGlyIle                              115120125                                                                     LeuHisArgAspLeuThrLeuSerAsnIleLeuLeuThrArgAsnMet                              130135140                                                                     AsnIleLysIleAlaAspPheGlyLeuAlaThrGlnLeuAsnMetPro                              145150155160                                                                  HisGluLysHisTyrThrLeuCysGlyThrProAsnTyrIleSerPro                              165170175                                                                     GluIleAlaThrArgSerAlaHisGlyLeuGluSerAspIleTrpSer                              180185190                                                                     LeuGlyCysMetSerTyrThrLeuLeuIleGlyArgProProPheAsp                              195200205                                                                     ThrAspThrValLysAsnThrLeuAsnLysValValLeuAlaAspTyr                              210215220                                                                     GluMetProAlaPheLeuSerArgGluAlaGlnAspLeuIleHisGln                              225230235240                                                                  LeuLeuArgArgAsnProAlaAspArgLeuSerLeuSerSerValLeu                              245250255                                                                     AspHisProPheMetSerArgAsnProSerProLysSerLysAspVal                              260265270                                                                     Gly                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 271 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Drosophila melanogaster                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      IleAsnGlnArgLysThrTyrLysArgMetArgPhePheGlyLysGly                              151015                                                                        GlyPheAlaLysCysTyrGluIleIleAspValGluThrAspAspVal                              202530                                                                        PheAlaGlyLysIleValSerLysLysLeuMetIleLysHisAsnGln                              354045                                                                        LysGluLysThrAlaGlnGluIleThrIleHisArgSerLeuAsnHis                              505560                                                                        ProAsnIleValLysPheHisAsnTyrPheGluAspSerGlnAsnIle                              65707580                                                                      TyrIleValLeuGluLeuCysLysLysArgSerMetMetGluLeuHis                              859095                                                                        LysArgArgLysSerIleThrGluPheGluCysArgTyrTyrIleTyr                              100105110                                                                     GlnIleIleGlnGlyValLysTyrLeuHisAspAsnArgIleIleHis                              115120125                                                                     ArgAspLeuLysLeuGlyAsnLeuPheLeuAsnAspLeuLeuHisVal                              130135140                                                                     LysIleGlyAspPheGlyLeuAlaThrArgIleGluTyrGluGlyGlu                              145150155160                                                                  ArgLysLysThrLeuCysGlyThrAlaAsnTyrIleAlaProGluIle                              165170175                                                                     LeuThrLysLysGlyHisSerPheGluValAspIleTrpSerIleGly                              180185190                                                                     CysValMetTyrThrLeuLeuValGlyGlnProProPheGluThrLys                              195200205                                                                     ThrLeuLysAspThrTyrSerLysIleLysLysCysGluTyrArgVal                              210215220                                                                     ProSerTyrLeuArgLysProAlaAlaAspMetValIleAlaMetLeu                              225230235240                                                                  GlnProAsnProGluSerArgProAlaIleGlyGlnLeuLeuAsnPhe                              245250255                                                                     GluPheLeuLysGlySerLysValProMetPheLeuProSerSer                                 260265270                                                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AspProThrThrGlyLysArgTyrCysArgGlyLysValLeuGlyLys                              151015                                                                        GlyGlyPheAlaLysCysTyrGluMetThrAspLeuThrAsnAsnLys                              202530                                                                        ValTyrAlaAlaLysIleIleProHisSerArgValAlaLysProHis                              354045                                                                        GlnArgGluLysIleAspLysGluIleGluLeuHisArgLeuLeuHis                              505560                                                                        HisLysHisValValGlnPheTyrHisTyrPheGluAspLysGluAsn                              65707580                                                                      IleTyrIleLeuLeuGluTyrCysSerArgArgSerMetAlaHisIle                              859095                                                                        LeuLysAlaArgLysValLeuThrGluProGluValArgTyrTyrLeu                              100105110                                                                     ArgGlnIleValSerGlyLeuLysTyrLeuHisGluGlnGluIleLeu                              115120125                                                                     HisArgAspLeuLysLeuGlyAsnPhePheIleAsnGluAlaMetGlu                              130135140                                                                     LeuLysValGlyAspPheGlyLeuAlaAlaArgLeuGluProLeuGlu                              145150155160                                                                  HisArgArgArgThrIleCysGlyThrProAsnTyrLeuSerProGlu                              165170175                                                                     ValLeuAsnLysGlnGlyHisGlyCysGluSerAspIleTrpAlaLeu                              180185190                                                                     GlyCysValMetTyrThrMetLeuLeuGlyArgProProPheGluThr                              195200205                                                                     ThrAsnLeuLysGluThrTyrArgCysIleArgGluAlaArgTyrThr                              210215220                                                                     MetProSerSerLeuLeuAlaProAlaLysHisLeuIleAlaSerMet                              225230235240                                                                  LeuSerLysAsnProGluAspArgProSerLeuAspAspIleIleArg                              245250255                                                                     HisAspPhePheLeuGlnGlyPheThrProAspArgLeuSerSerSer                              260265270                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 275 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Saccharomyces cerevisiae                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      IleLysThrArgGlyLysAspTyrHisArgGlyHisPheLeuGlyGlu                              151015                                                                        GlyGlyPheAlaArgCysPheGlnIleLysAspAspSerGlyGluIle                              202530                                                                        PheAlaAlaLysThrValAlaLysAlaSerIleLysSerGluLysThr                              354045                                                                        ArgLysLysLeuLeuSerGluIleGlnIleHisLysSerMetSerHis                              505560                                                                        ProAsnIleValGlnPheIleAspCysPheGluAspAspSerAsnVal                              65707580                                                                      TyrIleLeuLeuGluIleCysProAsnGlySerLeuMetGluLeuLeu                              859095                                                                        LysArgArgLysValLeuThrGluProGluValArgPhePheThrThr                              100105110                                                                     GlnIleCysGlyAlaIleLysTyrMetHisSerArgArgValIleHis                              115120125                                                                     ArgAspLeuLysLeuGlyAsnIlePhePheAspSerAsnTyrAsnLeu                              130135140                                                                     LysIleGlyAspPheGlyLeuAlaAlaValLeuAlaAsnGluSerGlu                              145150155160                                                                  ArgLysTyrThrIleCysGlyThrProAsnTyrIleAlaProGluVal                              165170175                                                                     LeuMetGlyLysHisSerGlyHisSerPheGluValAspIleTrpSer                              180185190                                                                     LeuGlyValMetLeuTyrAlaLeuLeuIleGlyLysProProPheGln                              195200205                                                                     AlaArgAspValAsnThrIleTyrGluArgIleLysCysArgAspPhe                              210215220                                                                     SerPheProArgAspLysProIleSerAspGluGlyLysIleLeuIle                              225230235240                                                                  ArgAspIleLeuSerLeuAspProIleGluArgProSerLeuThrGlu                              245250255                                                                     IleMetAspTyrValTrpPheArgGlyThrPheProProSerIlePro                              260265270                                                                     SerThrVal                                                                     275                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 272 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Mus musculus                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AspProArgSerArgArgGlnTyrIleArgGlyArgPheLeuGlyLys                              151015                                                                        GlyGlyPheAlaLysCysPheGluIleSerAspAlaAspThrLysGlu                              202530                                                                        ValPheAlaGlyLysIleValProLysSerLeuLeuLeuLysProHis                              354045                                                                        GlnLysGluLysMetSerMetGluIleSerIleHisArgSerLeuAla                              505560                                                                        HisGlnHisValValGlyPheHisAspPhePheGluAspSerAspPhe                              65707580                                                                      ValPheValValLeuGluLeuCysArgArgArgSerLeuLeuGluLeu                              859095                                                                        HisLysArgArgLysAlaLeuThrGluProGluAlaArgTyrTyrLeu                              100105110                                                                     ArgGlnIleValLeuGlyCysGlnTyrLeuHisArgAsnGlnValIle                              115120125                                                                     HisArgAspLeuLysLeuGlyAsnLeuPheLeuAsnGluAspLeuGlu                              130135140                                                                     ValLysIleGlyAspPheGlyLeuAlaThrLysValGluTyrGluGly                              145150155160                                                                  GluArgLysLysThrLeuCysGlyThrProAsnTyrIleAlaProGlu                              165170175                                                                     ValLeuSerLysLysGlyHisSerPheGluValAspValTrpSerIle                              180185190                                                                     GlyCysIleMetTyrThrLeuLeuValGlyLysProProPheGluThr                              195200205                                                                     SerCysLeuLysGluThrTyrLeuArgIleLysLysAsnGluTyrSer                              210215220                                                                     IleProLysHisIleAsnProValAlaAlaSerLeuIleGlnLysMet                              225230235240                                                                  LeuGlnThrAspProThrAlaArgProThrIleHisGluLeuLeuAsn                              245250255                                                                     AspGluPhePheThrSerGlyTyrIleProAlaArgLeuProIleThr                              260265270                                                                     __________________________________________________________________________

We claim:
 1. A purified and isolated nucleic acid molecule comprising anucleic acid sequence encoding a protein having the amino acid sequencefrom amino acids 5 to 277 as shown in SEQ ID NO:2.
 2. A purified andisolated nucleic acid molecule as claimed in claim 1 comprising anucleic acid sequence corresponding to nucleic acids 218 to 1036 asshown in SEQ ID NO:1, wherein T can also be U.
 3. A recombinant moleculeadapted for transformation of a host cell comprising a nucleic acidmolecule as claimed in claim 2 and one or more transcription andtranslation elements operatively linked to the nucleic acid molecule. 4.A purified and isolated nucleic acid molecule as claimed in claim 1comprising a nucleic acid sequence encoding a protein having the aminoacid sequence as shown in SEQ ID NO:4.
 5. A recombinant molecule adaptedfor transformation of a host cell comprising a nucleic acid molecule asclaimed in claim 4 and one or more transcription and translationelements operatively linked to the nucleic acid molecule.
 6. A purifiedand isolated nucleic acid molecule as claimed in claim 1 comprising anucleic acid sequence encoding a protein having the amino acid sequenceas shown in SEQ ID NO:6.
 7. A recombinant molecule adapted fortransformation of a host cell comprising a nucleic acid molecule asclaimed in claim 6 and one or more transcription and translationelements operatively linked to the nucleic acid molecule.
 8. Recombinantmolecules adapted for transformation of a host cell comprising a nucleicacid molecule as claimed in claim 1 and one or more transcription andtranslation elements operatively linked to the nucleic acid molecule. 9.A host cell containing a recombinant molecule as claimed in claims 8, 3,5 or
 7. 10. A method for producing a serine/threonine kinase proteincomprising transforming a host cell with a recombinant molecule asclaimed in claims 8, 3, 5, or 7 expressing the protein in a host cell;and, isolating the protein.
 11. A purified and isolated serine/threoninetyrosine kinase protein which is associated with mitotic and meioticcell division comprising the amino acid sequence as shown in SEQ ID.NO:2.
 12. A purified and isolated nucleic acid molecule which iscomplementary to the full length nucleic acid sequence corresponding tonucleic acids 218 to 1036 as shown in SEQ ID NO:1.
 13. A purified andisolated nucleic acid molecule consisting of a sequence encoding aminoacids 5 to 277 as shown in SEQ ID NO:2.
 14. A purified and isolatednucleic acid molecule consisting of a sequence encoding amino acids 417to 916 as shown in SEQ ID NO:4.
 15. A purified and isolated nucleic acidmolecule consisting of a sequence encoding amino acids 417 to 465 asshown in SEQ ID NO:6.
 16. A purified and isolated protein consisting ofamino acids 5 to 277 as shown in SEQ ID NO:2.
 17. A purified andisolated protein consisting of amino acids 417 to 916 as shown in SEQ IDNO:4.
 18. A purified and isolated protein consisting of amino acids 417to 465 as shown in SEQ ID NO:6.
 19. A purified and isolatedserine/threonine tyrosine kinase protein comprising the amino acidsequence as shown in SEQ ID NO:4.
 20. A purified and isolatedserine/threonine tyrosine kinase protein comprising the amino acidsequence as shown in SEQ ID NO:6.